Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.     

Identification of the 16 degrees C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells

In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16 degrees C. In virus-infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic reticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of approximately 2 but was apparent only at temperatures greater than 12 degrees C. A similar response was seen in situ at 12 degrees C and 16 degrees C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18 degrees C and below and especially at 8 degrees C and 12 degrees C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20 degrees C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37 degrees C were of a greater diameter than those formed at 4 degrees C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16 degrees C compartment observed in virally-infected cell lines grown at low temperatures.     

Low temperature compartment formation in feline immunodeficiency virus-infected and uninfected feline kidney cells

Summary This study was to determine if feline immunodeficiency virus (FIV)-infected and uninfected Crandall feline kidney (CRFK) cells exhibited a low temperature (16°C) block in membrane trafficking between transitional endoplasmic reticulum and Golgi apparatus represented by intermediate compartment formation. Cells were cultured at different temperatures and membrane changes involving the Golgi apparatus and Golgi apparatus-associated membrane structures were monitored by electron microscopy and quantitated. With 30 min of incubation, membranes of the Golgi apparatus stack increased in amount at temperatures of 16°C and below compared to temperatures above 18°C. The increase was greatest along the major polarity axis as evidenced by an increased stack height. Neither the number of cisternae per stack nor the average stack diameter (width) was affected by temperature. The response was maximal between 15 and 30 min of low temperature treatment of the cells. Results with cells infected and uninfected with feline immunodeficiency virus were similar. The increase in stack height was due primarily to an increase of membranes at the cis face (cis Golgi apparatus network). At 18°C, membranes of the trans Golgi apparatus network accumulated suggesting that import from the cis Golgi network could proceed at this temperature, whereas exit from the trans Golgi network was still at least partially blocked. Also increased at 16°C and below were numbers of transition vesicles in the space between the Golgi apparatus and the transitional endoplasmic reticulum associated with the cis Golgi apparatus face. The results suggested interruption of the orderly flux of membranes into the Golgi apparatus at 16°C and below. Moreover, the block appeared to be reversible. Upon transfer from 16°C to 37°C, there was a time-dependent decrease in the accumulations of cis compartment membrane accompanied by a corresponding equivalent increase in the membranes of the trans Golgi apparatus compartment.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-free transfer of membrane lipids. Evidence for lipid processing

A latent phospholipase A is concentrated in cis elements of rat liver Golgi apparatus, the presumed sites of fusion of the 50-70-nm transition vesicles formed from endoplasmic reticulum. As a result, conversion of transferred phospholipids to their corresponding lysoforms may provide an index of post transfer lipid processing in a corresponding reconstituted membrane transfer system. To label the phosphatidylcholine of transitional endoplasmic reticulum in vitro, [14C]CDP-choline and endogenous cytidyltransferases were used. In the reconstituted transfer system, the radiolabeled phosphatidylcholine was transferred via transition vesicles to Golgi apparatus immobilized on nitrocellulose strips in a time- and temperature-dependent process. Transfer was promoted by ATP and the ATP-dependent transfer was specific for cis Golgi apparatus elements as acceptor. Trans Golgi apparatus elements were ineffective as acceptors. Median Golgi apparatus elements were intermediate. A portion of the transferred phosphatidylcholine was converted subsequently to lysophosphatidylcholine also in a time- and ATP-dependent manner. The phospholipase A activity of the Golgi apparatus was more than 90% latent (active site located on the lumens of the Golgi apparatus membranes). Therefore, the lipid-containing vesicles derived from endoplasmic reticulum must have combined with cis Golgi apparatus membranes as the basis for Golgi apparatus-dependent phospholipase A processing of endoplasmic reticulum-derived phosphatidylcholine. Since the lipids were processed by phospholipase A in approximately the same proportion as occurs in situ, the findings offer evidence both for the specificity of the ATP-dependent component of cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus and its fidelity to lipid transfer observed in vivo.     

Cell-free transfer and sorting of membrane lipids in spinach

Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [¹⁴C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.     

NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver.

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.     

Physical membrane displacement: Reconstitution in a cell-free system and relationship to cell growth+

Summary Physical membrane displacement is a process common to all forms of vesicle budding as well as cell enlargement and pleomorphic shape changes. Cell-free reconstitution of membrane budding has been achieved with transitional endoplasmic reticulum fractions from both plants and animals where 50 to 70 nm transition vesicles have been observed to bud from the part-rough, part-smooth membrane elements that define transitional endoplasmic reticulum. This budding phenomenon requires ATP, is facilitated by cytosol and guanine nucleotides, and is both time- and temperature-dependent. The transitional endoplasmic reticulum buds that form when concentrated by preparative free-flow electrophoresis will attach specifically to cis Golgi apparatus membranes immobilized on nitrocellulose as an acceptor compartment. Golgi apparatus membranes derived from the trans compartment do not serve as an efficient acceptor compartment. Transfer of the vesicles once formed is rapid, nearly complete and no longer dependent upon added ATP. Transfer shows a strict temperature dependency corresponding to that of the intact cell where at temperatures of 16°C or below, vesicles form but do not attach to cis Golgi whereas at temperatures of greater than 16°C, vesicles both form and fuse. The principle ATPase of transitional endoplasmic reticulum which may be involved in the budding process has been identified, characterized and isolated. A 38 kDa cis Golgi apparatus associated protein also has been identified as a potential candidate as a docking protein. Transfer between trans Golgi apparatus and the plasma membrane also has been studied by cell-free analysis. Here, transfer has been found to be stimulated by NADH or NADH plus ascorbate. The role of NADH is unknown but the ability of plant and Golgi apparatus to oxidize NADH is inhibited by brefeldin A, a compound known to block membrane trafficking even at the level of the trans Golgi network. NADH oxidase activity of plasma membranes also has been described and is inhibited as well by brefeldin. Recent observations suggest that brefeldin A may block both the formation of vesicles at the trans Golgi apparatus as well as auxin hormone-stimulated cell elongation in plants. This once again raises the possibility of whether or not plant cell elongation is obligatorily mediated by membrane input from the Golgi apparatus. The latter seems unlikely based on two additional lines of evidence. The first is that auxin-induced cell elongation in plants shows no sharp temperature transition over the range of 4 to 24°C, whereas production of secretory vesicles from the trans Golgi apparatus appears to be largely prevented at temperatures of 18°C or less. Secondly, the sodium selective ionophore, monensin, which effectively blocks the formation of functional secretory vesicles at the trans Golgi apparatus, is also largely without effect on auxin-induced cell elongation for periods of 4 h or longer. Taken together the findings suggest that the action of brefeldin A on vesicle budding at the Golgi apparatus and cell enlargement, are not directly correlated but may represent a common action of the drug on some constituent essential to membrane displacement mechanisms.Abbreviations BFA brefeldin A - IAA indole-3-acetic acid; 2, 4-D 2, 4-dichlorophenoxyacetic acid - NSF N-ethylmaleimide-sensitive factor Much of the information summarized in this report was presented as a plenary lecture at the XV International Botanical Congress Tokyo, Yokohama, Japan, August 28–September 3, 1993.     

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

Summary The process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with¹²⁵I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.Abbrevations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

Transition vesicle formationin vitro

Summary Isolated fractions enriched in transition elements derived from part rough—part smooth regions of endoplasmic reticulum of rat liver respondin vitro to ATP plus a concentrated fraction of cytoplasmic proteins by formation of ca. 60 nm vesicles with nap-like coats resembling those of transition vesicles of the intact cell. Similar vesicles are normally considered to function in the transfer of materials from endoplasmic reticulum to cis elements of the Golgi apparatus.     

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.     

ATP-dependent cell-free transfer of membrane lipids from nuclei to Golgi apparatus of germinating axes of garden pea

Summary ATP-dependent cell-free transfer of membrane constituents radiolabeled with [¹⁴C]acetate, primarily lipids, was demonstrated between isolated nuclei in suspension and purified Golgi apparatus immobilized on nitrocellulose strips prepared from garden pea (Pisum sativum) in the presence of pea cytosol. The ATP-dependent transfer correlated with the ability of the nuclear envelope to form 50–70 nm vesicles and blebs in an ATP-dependent manner. Specific transfer, transfer at 23°C minus transfer at 4°C, was approximately doubled by addition of ATP and was greater for peas germinated for 2 days than for peas germinated for 3 days. ATP plus cytosol-dependent transfer could not be demonstrated using radiolabeled pea nuclei as donor with purified endoplasmic reticulum, plasma membrane, nuclei, mitochondria or amyloplasts as acceptors. The results provide a second example, in addition to transfer between endoplasmic reticulum and Golgi apparatus, where ATP-and temperature-dependent transfer via 50–70 nm transition vesicles can be demonstrated in a cell-free system.     

Transition vesicles of the cis Golgi apparatus face of rat liver are increased by retinol

Golgi apparatus of livers of rats receiving 60 mg/100 g body weight all-trans retinol (vitamin A) in olive oil responded by a reproducible and significant increase both in the number of cisternae per Golgi apparatus stack and in the number of transition vesicles of the cis Golgi apparatus face compared to rats receiving olive oil alone as determined by quantitation from electron micrographs. These vesicles were identified by a simple, non-clathrin coat, a uniform diameter of about 60 nm and a location primarily in association with cis Golgi apparatus elements. They were distinct from clathrin-coated vesicles of the trans Golgi apparatus face which was unaffected by vitamin A treatment. Transition vesicles may be involved in the transfer of membrane materials to the Golgi apparatus from endoplasmic reticulum.     

Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment.

Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers.     

Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring- shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity- purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide- sensitive fusion protein.     

Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP- sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.     

Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicle and Golgi apparatus fractions of rat liver

Coated vesicles were isolated from rat liver in about 80% fraction purity as determined from electron microscopy and analyses of marker enzymes and compared with Golgi apparatus and other membrane fractions isolated in parallel. The fractions were enriched in NADH-monodehydroascorbate reductase, ascorbate oxidase and ascorbic acid. The NADH-monodehydroascorbate reductase and ascorbate oxidase of the Golgi apparatus and coated vesicles differed from that of the endoplasmic reticulum in being inhibited by the sodium selective ionophore, monensin, at physiological concentrations while these activities were stimulated by ethylenediaminetetraacetic acid in coated vesicles but not in Golgi apparatus. Activities of both coated vesicles and Golgi apparatus fractions depleted in the coat protein, clathrin, were activated by the addition of clathrin-rich supernatant fractions. The results are discussed in the context of monodehydroascorbate as an acceptor for electron transport-mediated transfer of electrons from NADH by coated vesicles as part of a possible mechanism to drive membrane translocations or to acidify the interiors of vesicles.     

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

This review summarizes the data describing the role of cellular microtubules in transportation of membrane vesicles — transport containers for secreted proteins or lipids. Most events of early vesicular transport in animal cells (from the endoplasmic reticulum to the Golgi apparatus and in the opposite recycling direction) are mediated by microtubules and microtubule motor proteins. Data on the role of dynein and kinesin in early vesicle transport remain controversial, probably because of the differentiated role of these proteins in the movements of vesicles or membrane tubules with various cargos and at different stages of secretion and retrograde transport. Microtubules and dynein motor protein are essential for maintaining a compact structure of the Golgi apparatus; moreover, there is a set of proteins that are essential for Golgi compactness. Dispersion of ribbon-like Golgi often occurs under physiological conditions in interphase cells. Golgi is localized in the leading part of crawling cultured fibroblasts, which also depends on microtubules and dynein. The Golgi apparatus creates its own system of microtubules by attracting γ-tubulin and some microtubule-associated proteins to membranes. Molecular mechanisms of binding microtubule-associated and motor proteins to membranes are very diverse, suggesting the possibility of regulation of Golgi interaction with microtubules during cell differentiation. To illustrate some statements, we present our own data showing that the cluster of vesicles induced by expression of constitutively active GTPase Sar1a[H79G] in cells is dispersed throughout the cell after microtubule disruption. Movement of vesicles in cells containing the intermediate compartment protein ERGIC53/LMANI was inhibited by inhibiting dynein. Inhibiting protein kinase LOSK/SLK prevented orientation of Golgi to the leading part of crawling cells, but the activity of dynein was not inhibited according to data on the movement of ERGIC53/LMANI-marked vesicles.