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.     

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N- acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans- Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.     

Novel type Arabidopsis thaliana H(+)-PPase is localized to the Golgi apparatus

Vacuolar H(+)-PPase, a membrane bound proton-translocating pyrophosphatase found in various species including plants, some protozoan and prokaryotes, has been demonstrated to be localized to the vacuolar membrane in plants. Using a GUS reporter system and a green fluorescent protein (GFP) fusion protein, we investigated the tissue distribution and the subcellular localization, respectively, of a novel type H(+)-PPase encoded by AVP2/AVPL1 identified in the Arabidopsis thaliana genome. We showed that AVP2/AVPL1 is highly expressed at the trichome and the filament of stamen. Furthermore, the fluorescence of GFP-tagged AVP2/AVPL1 showed small dot-like structures that were observed throughout the cytoplasm of various Arabidopsis cells under a fluorescent microscope. The distribution of this dot-like fluorescent pattern was apparently affected by a treatment with brefeldin A. Moreover, we demonstrated that most dot-like fluorescent structures colocalized with a Golgi resident protein. These findings suggest that this novel type H(+)-PPase resides on the Golgi apparatus rather than the vacuolar membrane.     

A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons

(Ctm)PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. In previous studies, we found that PrP molecules carrying mutations in the N-terminal signal peptide (L9R) and the transmembrane domain (3AV) were synthesized exclusively in the (Ctm)PrP form in transfected cell lines. To characterize the properties of (Ctm)PrP in a neuronal setting, we have utilized cerebellar granule neurons cultured from Tg(L9R-3AV) mice that developed a fatal neurodegenerative illness. We found that about half of the L9R-3AV PrP synthesized in these neurons represents (Ctm)PrP, with the rest being (Sec)PrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated. (Sec)PrP was localized on the surface of neuronal processes. Most surprisingly, (Ctm)PrP was concentrated in the Golgi apparatus, rather in the endoplasmic reticulum as it is in transfected cell lines. Our study is the first to analyze the properties of (Ctm)PrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.     

Phospholipase D2 is localized to the rims of the Golgi apparatus in mammalian cells

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid, a molecule known to have multiple physiological roles, including release of nascent secretory vesicles from the trans-Golgi network. In mammalian cells two forms of the enzyme, PLD1 and PLD2, have been described. We recently demonstrated that PLD1 is localized to the Golgi apparatus, nuclei, and to a lesser extent, plasma membrane. Due to its low abundance, the intracellular localization of PLD2 has been characterized only indirectly through overexpression of chimeric proteins. Using antibodies specific to PLD2, together with immunofluorescence microscopy, herein we demonstrate that a significant fraction of endogenous PLD2 localized to the perinuclear Golgi region and was also distributed throughout cells in dense cytoplasmic puncta; a fraction of which colocalized with caveolin-1 and the plasma membrane. On treatment with brefeldin A, PLD2 translocated into the nucleus in a manner similar to PLD1, suggesting a potential role in nuclear signaling. Most significantly, cryoimmunogold electron microscopy demonstrated that in pituitary GH(3) cells >90% of PLD2 present in the Golgi apparatus was localized to cisternal rims and peri-Golgi vesicles exclusively. The data are consistent with a model whereby PLD2 plays a role in Golgi vesicular transport.     

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.     

Trypanosoma cruzi: TcRAB7 protein is localized at the Golgi apparatus in epimastigotes

In mammalian cells, the Rab7 protein is a key element of late endocytic membrane traffic. Several results suggest that it is involved in the transport from early to late endosome or from late endosome to lysosome. We have previously characterized a Rab7 gene homologue (TcRAB7) in Trypanosoma cruzi. Now, using an affinity-purified antibody specific to TcRAB7 protein we have determined that it is localized at the Golgi apparatus of the parasite. Our results indicate that the T. cruzi Rab7 homologue may function in a different route than its counterparts in mammalian cells.     

Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins

Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.     

The Golgi apparatus: defining the identity of Golgi membranes

The Golgi apparatus is a stack of compartments that serves as a central junction for membrane traffic, with carriers moving through the stack as well as arriving from, and departing toward, many other destinations in the cell. This requires that the different compartments in the Golgi recruit from the cytosol a distinct set of proteins to mediate accurate membrane traffic. This recruitment appears to reflect recognition of small GTPases of the Rab and Arf family, or of lipid species such as PtdIns(4)P and diacylglycerol, which provide a unique "identity" for each compartment. Recent work is starting to reveal the mechanisms by which these labile landmarks are generated in a spatially restricted manner on specific parts of the Golgi.     

Targeting of proteins to the Golgi apparatus

 The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure. Accepted: 15 October 1997     

Targeting of proteins to the Golgi apparatus

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recyclingtrans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.     

Deciphering the Golgi apparatus: from imaging to genes

The Golgi apparatus is a vital organelle in eukaryotic cells. It grabs and processes secretory materials synthesized by the endoplasmic reticulum (ER) before sorting them to their destination. The Golgi also receives materials from vacuoles/lysosomes and the plasma membrane for further recycling to other compartments within the cell (1) (Figure 1). Given the vital role of the Golgi in a cell, it is important to understand how this organelle attains and maintains its structural and functional integrity during the intense processes of membrane traffic. Despite an equally central role of the Golgi in membrane traffic in eukaryotes, the organization of this organelle has some unique features in each cell system. Therefore, the wealth of information available on the structure and activity of the Golgi in one system is not always directly transferable to others. However, certain morphological and functional aspects are common among cell systems. Therefore, studying the factors that regulate organelle biogenesis and organization of the Golgi apparatus is important in basic cell biology of eukaryotes and may also contribute to a better understanding of how different cell systems have evolved. In this study, we report on the identification of Golgi mutants in plant cells. We have developed a screen that is a promising strategy not only for the identification of genes responsible for the morphological and functional integrity of the plant Golgi but could also provide fundamental information on other multicellular systems for which the power of forward genetics cannot be exploited as easily as in Arabidopsis.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.     

Early effects of aminonucleoside on enzymes in the Golgi apparatus of rat liver.

Rats were injected with a single intravenous dose of aminonucleoside (AMN) and sacrificed 1-48 h later. The activity of several enzymes was assayed in the Golgi apparatus isolated from the liver. Galactosyltransferase activity showed little changes after the AMN, but both acid (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) activities increased within the first hour and reached control levels only 5-24 h later. Thiamine pyrophosphatase and arylsulfatase A (EC 3.1.6.1) activities also increased and stayed at higher levels for the duration of the experiment. Arylsulfatase B (EC 3.1.6.1) activity decreased shortly after the AMN but later increased to above control levels. These findings support earlier results in which liver ultrastructural and biochemical changes were observed early before renal lesions and proteinuria.     

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.     

The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations

The acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus.     

Topology-based abstraction of complex biological systems: application to the Golgi apparatus

Many complex cellular processes involve major changes in topology and geometry. We have developed a method using topology-based geometric modelling in which the edge labels of an n-dimensional generalized map (a subclass of graphs) represent the relations between neighbouring biological compartments. We illustrate our method using two topological models of the Golgi apparatus. These models can be animated using transformation rules, which depend on geometric and/or biochemical data and which modify both these data and the topology. Both models constitute plausible topological representations of the Golgi apparatus, but only the model based on a recent hypothesis about the Golgi apparatus is fully compatible with data from electron microscopy. Finally, we outline how our method may help biologists to choose between different hypotheses.