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.     

Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport

Frozen, thin sections of baby hamster kidney (BHK) cells were incubated with either concanavalin A (Con A) or Ricinus communis agglutinin I (RCA) to localize specific oligosaccharide moieties in endoplasmic reticulum (ER) and Golgi membranes. These lectins were then visualized using an anti-lectin antibody followed by protein A conjugated to colloidal gold. All Golgi cisternae and all ER membranes were uniformly labeled by Con A. In contrast, RCA gave a uniform labeling of only half to three-quarters of those cisternae on the trans side of the Golgi stack; one or two cis Golgi cisternae and all ER membranes were essentially unlabeled. This pattern of lectin labeling was not affected by infection of the cells with Semliki Forest virus (SFV). Infected cells transport only viral spike glycoproteins from their site of synthesis in the ER to the cell surface via the stacks of Golgi cisternae where many of the simple oligosaccharids on the spike proteins are converted to complex ones (Green, J., G. Griffiths, D. Louvard, P. Quinn, and G. Warren. 1981. J. Mol. Biol. 152:663-698). It is these complex oligosaccharides that were shown, by immunoblotting experiments, to be specifically recognized by RCA. Loss of spike proteins from Golgi cisternae after cycloheximide treatment (Green et al.) was accompanied by a 50% decrease in the level of RCA binding. Hence, about half of the RCA bound to Golgi membranes in thin sections was bound to spike proteins bearing complex oligosaccharides and these were restricted to the trans part of the Golgi stack. Our results strongly suggest that complex oligosaccharides are constructed in trans Golgi cisternae and that the overall movement of spike proteins is from the cis to the trans side of the Golgi stack.     

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.     

Tomographic evidence for continuous turnover of Golgi cisternae in Pichia pastoris

The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking. The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack.     

Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack

Using monoclonal antibodies and electron microscopy, we have localized N-acetylglucosamine transferase I within the Golgi apparatus. This enzyme initiates the conversion of asparagine-linked oligosaccharides to the complex type. We have found that the enzyme is concentrated in the central (or medial) cisternae of the Golgi stack. Cisternae at the cis and trans ends of the Golgi complex appear to lack this protein. These experiments establish a function for the medial portion of the Golgi and imply that the Golgi is partitioned into at least three biochemically and morphologically distinct cisternal compartments.     

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.     

The Golgi apparatus ofPhytophthora cinnamomi makes three types of secretory or storage vesicles concurrently

Summary The formation of three types of vesicles in the oomycetePhytophthora cinnamomi was investigated using ultrastructural and immunocytochemical techniques. All three vesicles are synthesised at the same time; one type serves a storage role; the others undergo regulated secretion. A monoclonal antibody Lpv-1 that is specific for glycoproteins contained in the storage vesicles labelled the endoplasmic reticulum (ER), elements in the transition region between ER and Golgi stack, and cis, medial and trans Golgi cisternae. Cpa2, a monoclonal antibody specific for glycoproteins contained within secretory dorsal vesicles labelled the transition region, cis cisternae and a trans-Golgi network. Vesicles possessing a structure characteristic of mature secretory ventral vesicles were observed in close association with the trans face of Golgi stacks. The results suggest that all three vesicles are formed by the Golgi apparatus. Double immunogold labelling with Lpv-1 and Cpa-2 showed that these two sets of glycoproteins occurred within the same Golgi cisternae, indicating that both products pass through and are sorted concurrently within a single Golgi stack.     

Detection of glycosaminoglycans in the Golgi complex of chondrocytes

Elongation and sulfation of glycosaminoglycans are pivotal roles of the Golgi complex during the biosynthesis of proteoglycan monomers. In the present work the spatial relationship between these processes has been investigated by using a combination of immunocytochemical and cytochemical techniques. Chondroitin sulfate and keratan sulfate glycosaminoglycans were immunocytochemically localized in 1 to 2 transmost cisternae, also in a system of narrow tubules at the trans face of the Golgi complex of chick epiphyseal chondrocytes. At these same locations sulfate groups were revealed with the high iron diamine (HID) method, proteoglycan monomers being visualized with ruthenium red. Several treatments were assayed in order to reversibly block the secretory pathway. Chondrocytes incubated at a low temperature, 15 degrees C, before fixation, showed both glycosaminoglycans in the middle cisternae of the Golgi stack as well as the above mentioned locations. After low temperature treatment both HID and ruthenium red stained the middle, but not the cis cisternae. Incubation of the cells for 30 min with either diethylcarbamazine or monensin before fixation permitted detection of glycosaminoglycans and proteoglycan monomers in the middle cisternae, whereas HID staining of the Golgi complex, but not that of secretory vesicles, was abolished. The results show that elongation of both chondroitin sulfate and keratan sulfate glycosaminoglycans takes place in the same Golgi compartments. These include the middle cisternae and probably also the trans cisternae and tubules. Also suggested is that sulfation of one or both types of glycosaminoglycans begins in the middle cisternae.     

Transport of horseradish peroxidase from the cell surface to the Golgi in insulin-secreting cells: preferential labelling of cisternae located in an intermediate position in the stack.

We have used serial sectioning to study the topology of Golgi cisternae in insulin-secreting cells during secretion-stimulated endocytotic uptake of exogenous horseradish peroxidase (HRP). HRP-labelled cisternae were followed on several series of consecutive sections. This revealed that labelled cisternae could always be traced to a position in the Golgi stack intermediate between the cis and the trans poles. This occurred in spite of the apparent cis or trans locations of HRP-containing cisternae on some sections. The latter images could be explained by the lack of the true cis or trans (clathrin-coated) cisternae at certain levels of the stack.     

Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins.

Chromogranin B and secretogranin II, two members of the granin family, are known to be post-translationally modified by the addition of O-linked carbohydrates to serine and/or threonine, phosphate to serine and threonine, and sulfate to carbohydrate and tyrosine residues. In the present study, chromogranin B and secretogranin II were used as model proteins to investigate in which subcompartment of the Golgi complex secretory proteins become phosphorylated. Monensin, a drug known to block the transport from the medial to the trans cisternae of the Golgi stack, inhibited the phosphorylation of the granins, indicating that this modification occurred distal to the medial Golgi. Monensin also blocked the addition of galactose to O-linked carbohydrates and the sulfation of the granins, confirming previous data that these modifications take place in the trans Golgi. To distinguish, within the trans Golgi, between the trans cisternae of the Golgi stack and the trans Golgi network, we made use of the previous observation that brefeldin A results in the redistribution to the endoplasmic reticulum of membrane-bound enzymes of the trans cisternae of the Golgi stack, but not of the trans Golgi network. Brefeldin A treatment abolished granin sulfation but resulted in the accumulation of phosphorylated and galactosylated granins. Differential effects of brefeldin A on membranes of the Golgi stack versus the trans Golgi network were also observed by immunofluorescence analysis of marker proteins specific for either compartment. Our results suggest that the phosphorylation of secretory proteins, like their galactosylation, largely occurs in the trans cisternae of the Golgi stack, whereas the sulfation of secretory proteins on both carbohydrate and tyrosine residues takes place selectively in the trans Golgi network.     

The role of ARF1 and rab GTPases in polarization of the Golgi stack

The organization and sorting of proteins within the Golgi stack to establish and maintain its cis to trans polarization remains an enigma. The function of Golgi compartments involves coat assemblages that facilitate vesicle traffic, Rab-tether-SNAP receptor (SNARE) machineries that dictate membrane identity, as well as matrix components that maintain structure. We have investigated how the Golgi complex achieves compartmentalization in response to a key component of the coat complex I (COPI) coat assembly pathway, the ARF1 GTPase, in relationship to GTPases-regulating endoplasmic reticulum (ER) exit (Sar1) and targeting fusion (Rab1). Following collapse of the Golgi into the ER in response to inhibition of activation of ARF1 by Brefeldin A, we found that Sar1- and Rab1-dependent Golgi reformation took place at multiple peripheral and perinuclear ER exit sites. These rapidly converged into immature Golgi that appeared as onion-like structures composed of multiple concentrically arrayed cisternae of mixed enzyme composition. During clustering to the perinuclear region, Golgi enzymes were sorted to achieve the degree of polarization within the stack found in mature Golgi. Surprisingly, we found that sorting of Golgi enzymes into their subcompartments was insensitive to the dominant negative GTP-restricted ARF1 mutant, a potent inhibitor of COPI coat disassembly and vesicular traffic. We suggest that a COPI-independent, Rab-dependent mechanism is involved in the rapid reorganization of resident enzymes within the Golgi stack following synchronized release from the ER, suggesting an important role for Rab hubs in directing Golgi polarization.     

Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells

The recycling itinerary of plasma membrane transferrin receptors (TFR) was charted in IgG-secreting mouse myeloma cells (RPC 5.4) by tagging surface receptors with either bound anti-transferrin receptor antibodies (anti-TFR) or Fab fragments thereof and determining the intracellular destinations of the tagged receptors by immunocytochemistry. By immunofluorescence, TFR tagged with either probe were seen to be rapidly internalized and translocated from the cell surface to the juxtanuclear (Golgi) region. When localized by immunoperoxidase procedures at the electron microscopic level, the anti-TFR-labeled receptors were detected in all cisternae (cis, middle, and trans) of the Golgi stacks as well as in endosomes and trans Golgi reticular elements. There was no difference in the routing of TFR tagged with monovalent Fab and those tagged with divalent IgG. Tagged receptors were detected in Golgi stacks of approximately 50% of the cells analyzed. The position of the labeled cisternae within a given stack was found to be quite variable with cis and middle cisternae more often labeled at 5 min and trans cisternae at 30 min of antibody uptake. The finding that recycling plasmalemmal TFR can visit all or most Golgi subcompartments raises the likely possibility that any Golgi-associated posttranslational modification can occur during recycling as well as during the initial biosynthesis of plasmalemma receptors and other membrane proteins.     

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.     

RCA I-binding patterns of the Golgi apparatus

The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.     

Golgi structure in three dimensions: functional insights from the normal rat kidney cell

Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.     

Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface

The effect of reduced temperature on synchronized transport of SFV membrane proteins from the ER via the Golgi complex to the surface of BHK-21 cells revealed two membrane compartments where transport could be arrested. At 15 degrees C the proteins could leave the ER but failed to enter the Golgi cisternae and accumulated in pre-Golgi vacuolar elements. At 20 degrees C the proteins passed through Golgi stacks but accumulated in trans-Golgi cisternae, vacuoles, and vesicular elements because of a block affecting a distal stage in transport. Both blocks were reversible, allowing study of the synchronous passage of viral membrane proteins through the Golgi complex at high resolution by immunolabeling in electron microscopy. We propose that membrane proteins enter the Golgi stack via tubular extensions of the pre-Golgi vacuolar elements which generate the Golgi cisternae. The proteins pass across the Golgi apparatus following cisternal progression and enter the post-Golgi vacuolar elements to be routed to the cell surface.     

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes

Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternae of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.     

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.     

Structure of Golgi apparatus

Summary Golgi apparatus (GA) of eukaryotic cells consist of one or more stacks of flattened saccules (cisternae) and an array of fenestrae and tubules continuous with the peripheral edges of the saccules. Golgi apparatus also are characterized by zones of exclusion that surround each stack and by an assortment of vesicles (or vesicle buds) associated with both the stacks and the peripheral tubules of the stack cisternae. Each stack (sometimes referred to as Golgi apparatus, Golgi complex, or dictyosome) is structurally and functionally polarized, reflecting its role as an intermediate between the endoplasmic reticulum, the cell surface, and the lysosomal system of the cell. There is probably only one GA per cell, and all stacks of the GA appear to function synchronously. All Golgi apparatus are involved in the generation and movement of product and membrane within the cell or to the cell exterior, and these functions are often reflected as structural changes across the stacks. For example, in plants, both product and membrane appear to maturate from the cis to the trans poles of the stacks in a sequential, or serial, manner. However, there is also strong ultrastructural evidence in plants for a parallel input to the stack saccules, probably through the peripheral tubules. The same modes of functioning probably also occur in animal GA; although here, the parallel mode of functioning almost surely predominates. In some cells at least, GA stacks give rise to tubular-vesicular structures that resemble the trans Golgi network. Rudimentary GA, consisting of tubular-vesicular networks, have been identified in fungi and may represent an early stage of GA evolution.