Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.     

Two integral membrane proteins located in the cis-middle and trans-part of the Golgi system acquire sialylated N-linked carbohydrates and display different turnovers and sensitivity to cAMP-dependent phosphorylation

The localization and chemical characteristics of two Golgi integral membrane proteins (GIMPs) have been studied using monoclonal antibodies. The two proteins are segregated in different parts of the Golgi system and whereas GIMPc(130 kD) is located in the cis and medial cisternae, GIMPt (100 kD) is confined in the trans-most cisterna and trans-tubular network. Both GIMPs are glycoproteins that contain N- and O-linked carbohydrates. The N-linked carbohydrates were exclusively of the complex type. Although excluded from the trans-side of the Golgi system, where sialylation is believed to occur, GIMPc acquires sialic acid in both its N- and O-linked carbohydrates. Sialic acid was also detected in the N-linked carbohydrates of GIMPt. GIMPc is apparently phosphorylated in the luminal domain in vivo. Phosphorylation occurred exclusively on serine and was stimulated by dibutyryl cyclic AMP. GIMPc and GIMPt displayed half-lives of 20 and 9 h, respectively.     

Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.     

人分泌粒蛋白III的克隆和表达(英文)

研究了一个新的人分泌蛋白基因_分泌粒蛋白III(secretograninIII,SgIII)。SgIII蛋白序列共有 4 6 8个氨基酸残基 ,N端有一段疏水信号肽 ,序列中含有DSTK重复序列和 7对二元碱性氨基酸 (dibasicsites) ,这些结构特点同其他分泌粒蛋白家族成员相类似。人源SgIII蛋白在小鼠、大鼠和爪蟾中各有一个同源蛋白。基因组分析表明 ,SgIII基因位于人 15号染色体上 ,含有 12个外显子 ,分布在 39kb长的基因组DNA上。Western印迹和免疫细胞化学实验证实 ,SgIII蛋白同其他分泌粒蛋白家族成员一样 ,通过分泌途径被分泌到胞外。SgIII在多种组织中都有表达 ,Northern印迹显示SgIII的mRNA主要有 2 .2kb和 1.9kb两种形式 ,但在脑中还有 4 .5kb和 3.3kb大小的两种特异转录本。     

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.     

Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.     

Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein alpha(1)-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30-70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas alpha(1)-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.     

The Neuroendocrine Proteins Secretogranin II and III Are Regionally Conserved and Coordinately Expressed with Proopiomelanocortin in Xenopus Intermediate Pituitary

Abstract: Chromogranins and secretogranins are acidic secretory proteins of unknown function that represent major constituents of neuroendocrine secretory granules. Using a differential screening strategy designed to identify genes involved in peptide hormone biosynthesis and secretion, we have isolated cDNA clones encoding the first nonmammalian homologues of secretogranin II (SgII) and secretogranin III (SgIII) from a Xenopus intermediate pituitary cDNA library. A comparative analysis of the Xenopus and mammalian proteins revealed a striking regional conservation with an overall sequence identity of 48% for SgII and 61% for SgIII. One of the highly conserved and thus potentially functional domains in SgII corresponds to the bioactive peptide secretoneurin. However, in SgII and especially in SgIII, a substantial portion of the potential dibasic cleavage sites is not conserved, arguing against the idea that these granins serve solely as peptide precursors. Moreover, SgIII contains a conserved and repeated motif (DSTK) that is reminiscent of a repeat present in the trans -Golgi network integral membrane proteins TGN38 and TGN41, a finding more consistent with an intracellular function for this protein. When Xenopus intermediate pituitary cells were stimulated in vivo, the mRNA levels of SgII and SgIII increased dramatically (15- and 35-fold, respectively) and in parallel with that of the prohormone proopiomelanocortin (30-fold increase). Our results indicate that the process of peptide hormone production and release in a neuroendocrine cell involves multiple members of the granin family.     

In vitro transport on cis and trans sides of the Golgi involves two distinct types of coatomer and ADP-ribosylation factor-independent transport intermediates

The cisternal maturation model proposes that secretory proteins transit the Golgi in cisternae that mature by the continuous retrograde transport of Golgi enzymes in vesicles. We have tested the hypothesis that de novo generation of transport intermediates containing medial, trans, and trans Golgi network (TGN) enzymes is reconstituted in vitro. Our analysis shows that the majority of transport is mediated by a steady state of transport intermediate production and consumption by Golgi cisternae, with only a minor contribution of pre-existing transport intermediates. Transport in the medial and trans regions of the stack involved intermediates containing Golgi enzymes, apparently moving in a retrograde direction. In contrast, transport between the trans Golgi and TGN was exclusively mediated by intermediates containing secretory protein, as expected for anterograde transport. These intermediates may be physiologically relevant, because only these two specific types of intermediates can be detected in cell homogenates. By analogy to the coatomer (COPI)-independent transport of Golgi enzymes to the endoplasmic reticulum, the steady-state production of intra-Golgi transport intermediates was not impaired by inhibition of COPI vesicle formation. These data suggest a model for COPI-independent intra-Golgi transport by cisternal maturation with a shift in mechanism to anterograde transport at the trans Golgi and TGN boundary.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells

Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the Golgi apparatus is a subject of intense research. The protein kinase D (PKD) family of serine threonine kinases has so far been implicated in the regulation of constitutive secretion in epithelial cells. Here we examined whether PKD2 expression and activity could also play a role in the release of secretory granules from the trans Golgi network (TGN) in neuroendocrine tumour cells and hence be a target to block autonomous secretion by these tumours. Our data show that expression and catalytic activity of PKD2 are required for the release of chromogranin A containing secretory vesicles. Inhibition of PKD2 activity or siRNA knockdown of PKD2 resulted in a marked perinuclear retention of chromogranin A immunofluorescence in the trans Golgi network and led to a marked reduction in basal as well as phorbol ester stimulated secretion of chromogranin A into the supernatant of cells. Thus, PKD2 controls the release of secretory granules in neuroendocrine tumour cells at the level of the Golgi apparatus and could hence serve as a novel target to block hormone secretion in functional neuroendocrine tumours.     

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.     

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy

 Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for α1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable troughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis. Accepted: 12 January 1998     

Inhibition of Intracellular Pectin Transport in Pollen Tubes by Monensin,Brefeldin A and Cytochalasin D*

Specific inhibitors of the secretory pathway represent important tools for investigation of cell wall synthesis and tip growth in pollen tubes. Brefeldin A completely inhibits germination of Nicotiana tabacum pollen tubes at 2.2 μM. Ultrastructural investigation of pollen tube cytoplasm showed that brefeldin A caused the appearance of reticular structures and “brefeldin A compartments” containing unesterified pectins. Monensin caused inhibition of pollen tube germination at 80 nM. The drug induced swelling of the Golgi cisternae, many of which contained methyl-esterified pectins. Cytochalasin D was effective at 1 μg/ml, the inhibition of germination being fully reversible. Application of the drug caused accumulation of secretory vesicles containing methyl-esterified pectin around the dictyosomes. In contrast to brefeldin A and monensin, cytochalasin D caused a slowdown of cytoplasmic streaming. Monensin, but not the other drugs, caused a considerable decrease in pollen tube diameter. The characterization and quantification of the effects of the drugs on pollen tubes represents a necessary prerequisite for their application in physiological studies.     

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.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Arachidonyltrifluoromethy ketone, a phospholipase A(2) antagonist, induces dispersal of both Golgi stack- and trans Golgi network-resident proteins throughout the cytoplasm

Arachidonyltrifluoromethy ketone (AACOCF(3)), a phospholipase A(2) antagonist, reversibly induced dispersal of Golgi stack- and trans Golgi network (TGN)-resident proteins throughout the cytoplasm in NRK cells as followed by immunocytochemical staining of ManII and TGN38, respectively. The action of AACOCF(3) was partly blocked by other PLA(2) antagonists, suggesting it be not caused by a general inhibition of phospholipase A(2). AACOCF(3) neither dissociated beta-COP from membranes nor prevented brefeldin A-induced beta-COP release. Action of AACOCF(3) on the Golgi stack and TGN is different from that of brefeldin A and nordihydroguaiaretic acid. The most prominent difference is that the Golgi stack and TGN showed a similar sensitivity to AACOCF(3), while the TGN was dispersed more slowly than the Golgi stack in brefeldin A- or nordihydroguaiaretic acid-treated NRK cells. This novel action of AACOCF(3) may be used as pharmacological tool and give new insights into vesicle-mediated traffic and Golgi membrane dynamics.     

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.     

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday.     

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.