Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.     

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucosaminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyltransferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.     

Evidence for inherent differences in the system A carrier from normal and transformed liver tissue. Differential inactivation and substrate protection in membrane vesicles and reconstituted proteoliposomes

Plasma membrane vesicles isolated from intact rat liver (normal hepatocyte) or cultured rat H4 hepatoma cells retain Na+-dependent uptake of 2-aminoisobutyric acid mediated by System A. The carrier was inactivated in normal liver membrane vesicles by either N-ethylmaleimide (NEM) or p-chloromercuribenzene sulfonate (PCMBS). The concentrations required to produce half-maximal inhibition were approximately 370 and 110 microM for NEM and PCMBS, respectively. In contrast, transport of System A in H4 hepatoma membrane vesicles was sensitive to PCMBS (K 1/2 = 180 microM), yet totally unaffected by NEM at concentrations up to 5 mM. Substrate-dependent protection from PCMBS activation was observed for the System A activity in H4 hepatoma membranes, but not in vesicles from normal hepatocytes. Subsequent inactivation of the substrate-protected carrier by sulfhydryl-specific reagents, added following the removal of the protective amino acid, suggests that one or more cysteine residues become less reactive in the presence of System A substrates. Treatment of solubilized membrane proteins with NEM prior to reconstitution into artificial proteoliposomes showed that the selective inactivation by NEM of the carrier in normal liver membranes is not dependent on the lipid environment or on the integrity of the plasma membrane. The results support the hypothesis that there are inherent differences in the System A carriers that are present in normal and transformed liver tissue.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER

In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.     

CMP-N-acetylneuraminic acid hydrolase, an ectoenzyme distributed unevenly over the hepatocyte surface.

The regional localization of CMP-N-acetylneuramic acid hydrolase at the hepatocyte surface was studied by using plasma membranes and hepatocytes isolated from rat liver. 1. By homogenization of the rat liver plasma membrane preparations and subsequent discontinuous sucrose gradient centrifugation, one light and two heavy membrane fractions were obtained. The origin of these three subfractions is discussed based on the specific activities in the three fractions of 5'-nucleotidase, alakaline phosphatase and Mg2+-ATPase and on electron microscopic examination of the fractions. Evidence is given suggesting that the light fraction is derived from the bile canalicular surface of the plasma membrane, and that the heavy fractions are derived predominantly from the sinusoidal and lateral surfaces of the liver cell membrane. CMP-AcNeu hydrolase was present at highest specific activity in one of the heavy subfractions. Therefore it is concluded that CMP-AcNeu hdyrolase is located preferentially in the sinusoidal and/or lateral plasma membrane parts of the liver cell. 2. Experiments with intact and disintegrated hepatocytes isolated from rat liver indicated that CMP-AcNeu hydrolase is located at the surface of the cell membrane, with its functional group directed to the outside.     

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.     

Passage of serum-destined proteins through the Golgi apparatus of rat liver. An examination of heavy and light Golgi fractions

The participation of hepatic Golgi apparatus in the intracellular transport of blood-destined proteins has been analyzed using Golgi fractions enriched in cis and trans components of the Golgi apparatus. SDS-polyacrylamide gel electrophoresis of the liver Golgi fractions showed several proteins corresponding in relative proportions and mobilities with serum proteins. After a pulse injection of labeled leucine, the secretory content of the cis Golgi fraction was labeled earlier than the trans Golgi fraction. Taken together, the results show the participation of the liver Golgi apparatus in the secretion of most of the serum proteins and provide documentation for a sequential progression of secretory protein through the cis and trans components of the Golgi apparatus.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.     

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.     

Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin

In the accompanying paper (Griffiths, G., P. Quinn, and G. Warren, 1983, J. Cell Biol., 96:835-850), we suggested that the Golgi stack could be divided into functionally distinct cis, medial, and trans compartments, each comprising one or two adjacent cisternae. These compartments were identified using Baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV) and treated with monensin. This drug blocked intracellular transport but not synthesis of the viral membrane proteins that were shown to accumulate in the medial cisternae. In consequence, these cisternae bound nucleocapsids. Here we show that this binding markedly increased the density of the medial cisternae and allowed us to separate them from cis and trans Golgi cisternae. A number of criteria were used to show that the intracellular capsid-binding membranes (ICBMs) observed in vivo were the same as those membranes sedimenting to a higher density in sucrose gradients in vitro, and this separation of cisternae was then used to investigate the distribution, within the Golgi stack, of some specific Golgi functions. After labeling for 2.5 min with [3H]palmitate, most of the fatty acid attached to viral membrane proteins was found in the ICBM fraction. Because the viral membrane proteins appear to move from cis to trans, this suggests that fatty acylation occurs in the cis or medial Golgi cisternae. In contrast, the distribution of alpha 1-2- mannosidase, an enzyme involved in trimming high-mannose oligosaccharides, and of galactosyl transferase, which is involved in the construction of complex oligosaccharides, was not affected by monensin treatment. Together with data in the accompanying paper, this would restrict these two Golgi functions to the trans cisternae. Our data strongly support the view that Golgi functions have specific and discrete locations within the Golgi stack.     

Subfractionation of rat liver Golgi apparatus by free-flow electrophoresis

Using the technique of preparative free-flow electrophoresis, cisternae of unstacked rat liver Golgi apparatus were separated into a series of fractions of increasing content of sialic acid, thiamine pyrophosphatase and 5'-nucleotidase, markers regarded as being concentrated toward the mature Golgi apparatus face. These same fractions showed a decreasing content of nucleoside diphosphatase, an endoplasmic reticulum marker. Fractions enriched in sialic acid also were enriched in cisternae from the mature or trans face of the Golgi apparatus as deduced from cytochemical criteria. Those fractions least enriched in sialic acid contained cisternae that accumulated deposits of reduced osmium under standard conditions, a test used to mark the opposite, forming or cis-face. Thus subfractionation along the functional polarity axis of the Golgi apparatus with separation of cis and trans face cisternae has been achieved.     

Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus

Baby hamster kidney (BHK) cells were infected with Semliki Forest virus (SFV) and, 2 h later, were treated for 4 h with 10 microM monensin. Each of the four to six flattened cisternae in the Golgi stack became swollen and separated from the others. Intracellular transport of the viral membrane proteins was almost completely inhibited, but their synthesis continued and they accumulated in the swollen Golgi cisternae before the monensin block. In consequence, these cisternae bound large numbers of viral nucleocapsids and were easily distinguished from other swollen cisternae such as those after the block. These intracellular capsid-binding membranes (ICBMs) were not stained by cytochemical markers for endoplasmic reticulum (ER) (glucose-6-phosphatase) or trans Golgi cisternae (thiamine pyrophosphatase, acid phosphatase) but were labeled by Ricinus communis agglutinin I (RCA) in thin, frozen sections. Since this lectin labels only Golgi cisternae in the middle and on the trans side of the stack (Griffiths, G., R. Brands, B. Burke, D. Louvard, and G. Warren, 1982, J. Cell Biol., 95:781-792), we conclude that ICBMs are derived from Golgi cisternae in the middle of the stack, which we term medial cisternae. The overall movement of viral membrane proteins appears to be from cis to trans Golgi cisternae (see reference above), so monensin would block movement from medial to the trans cisternae. It also blocked the trimming of the high-mannose oligosaccharides bound to the viral membrane proteins and their conversion to complex oligosaccharides. These functions presumably reside in trans Golgi cisternae. This is supported by data in the accompanying paper, in which we also show that fatty acids are covalently attached to the viral membrane proteins in the cis or medial cisternae. We suggest that the Golgi stack can be divided into three functionally distinct compartments, each comprising one or two cisternae. The viral membrane proteins, after leaving the ER, would all pass in sequence from the cis to the medial to the trans compartment.     

Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi-intact cells.

The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.     

Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.

We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.     

Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.     

Galactose transfer to endogenous acceptors within Golgi fractions of rat liver

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.