Behavior of a transitional tubulovesicular compartment at the cis side of the Golgi apparatus in in vivo fusion studies of mammalian cells

We have investigated the behavior in in vivo cell fusion experiments of a transitional compartment lying between the endoplasmic reticulum and Golgi apparatus to determine if the compartment, as recognized by the antibody G1/93, might congregate in a similar manner to Golgi apparatus [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315-327]. The distributions of the transitional tubulovesicular compartment, endoplasmic reticulum, and Golgi apparatus in HeLa cells were assessed by immunofluorescent staining using mouse monoclonal antibody G1/93, mouse monoclonal antibody HP 24, and rabbit anti-galactosyltransferase, respectively. In agreement with previous results [W. C. Ho et al. (1990) Eur. J. Cell Biol. 52, 315-327], the Golgi apparatus was observed to congregate gradually over a 3- to 6-h period, forming a large, extended, central Golgi complex in uv-inactivated Sindbis virus-fused HeLa cells. Concomitant with this was a marked congregation of the transitional tubulovesicular compartment. Congregation of the tubulovesicular compartment was not affected by cycloheximide. The endoplasmic reticulum retained its web-like distribution throughout the syncytoplasm and rimmed the nuclear periphery. Treatment of HeLa cells with nocodazole prior to fusion followed by incubation of the syncytia in drug-containing media blocked congregation of the G1/93-positive compartment. With this long-term nocodazole treatment, Golgi apparatus was dispersed into scattered Golgi elements and the G1/93 distribution was endoplasmic reticulum-like. These results suggest that the transitional tubulovesicular compartment recognized by G1/93 is normally structured on microtubules and microtubule organizing centers and may be considered to be a subcompartment of a greater, perinuclear, Golgi complex.     

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.     

Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.     

Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum

The Golgi apparatus forms the heart of the secretory pathway in eukaryotic cells where proteins are modified, processed and sorted. The transport of proteins from the endoplasmic reticulum (ER) to the cis- side of the Golgi complex takes place at specialized ER sub-domains known as transitional ER (tER). We used the Plasmodium falciparum orthologue of Sec13p to analyse tER organization. We show that the distribution of Pf Sec13p is restricted to defined areas of the ER membrane. These foci are juxtaposed to the Golgi apparatus and might represent tER sites. To further analyse cis - to trans -Golgi architecture, we generated a double transfectant parasite line that expresses the Golgi marker Golgi reassembly stacking protein (GRASP) as a green fluorescent protein fusion and the trans- Golgi marker Rab6 as a DsRed fusion protein. Our data demonstrate that Golgi multiplication is closely linked to tER multiplication, and that parasite maturation is accompanied by the spatial separation of the cis- and trans- face of this organelle.     

Influence of antimicrotubular drugs on the Golgi apparatus of stomach secretory mucoid cells and small intestine absorptive cells

The effects of vinblastine and colchicine on the Golgi apparatus of stomach surface mucoid and absorptive intestinal cells were compared by cytochemical analysis. The two epithelial cells were chosen because of their different specific functions in the formation of secretory granules, the production of lysosomes and the intensity of membrane traffic in the cytoplasm. For the analysis, adult mice were injected with 1 mg/100 g b.w. of vinblastine and 1 mg/100 g b.w. of colchicine. For the demonstration of cis and trans cisternae of the Golgi apparatus, prolonged osmification, thiamine pyrophosphatase and acid phosphatase activity identification were applied. After treatment with vinblastine or colchicine, polarity of stacks in the Golgi apparatus of surface mucoid cells is preserved although the number of cisternae with thiamine pyrophosphatase or acid phosphatase activity decreases. However, the Golgi apparatus of intestinal absorptive cells completely disintegrates and only a few separated cis or trans cisternae can be identified. The main effect seems to be a reduction of vesicles which can be cytochemically identified as parts of the Golgi apparatus and an accumulation of vesicles which probably originate from budding ER. Communication between the ER and the Golgi apparatus seems to be interrupted.     

Antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the Golgi apparatus

In this study we report the identification of an antibody in the sera of some patients with autoimmune disease that reacted with a cytoplasmic antigen localized within the Golgi apparatus. The antibody reacted with all tissues investigated, which included pancreas, kidney, testis, liver, thymus, and spleen. In addition, it reacted with some human peripheral circulating lymphocytes, murine peritoneal macrophages, and a variety of tissue culture cell lines, which included HEp-2 cells (human epithelial carcinoma), baby hamster kidney cells, a canine thymus cell line, a primary kidney cell line, Ehrlich ascites cells, Wil-2 cells, and Raji cells. The antigen is located in the same region stained by the histochemical reaction for thiamine pyrophosphatase, thus indicating that the antigen is located within the Golgi apparatus. The antigen was not demonstrated by immunodiffusion of saline extracts of rabbit thymus, pancreas, or liver. The antigen in HEp-2 cells was resistant to RNase A, DNase I, micrococcal nuclease, and to extraction with 0.1 N HC1, but was sensitive to trypsin and Proteinase K. Eight patients with anti-Golgi antibodies have been identified. Six of the eight had systemic lupus erythematosus. Autoantibodies to a Golgi apparatus antigen might serve as a useful biologic marker to study the functional relationship of the Golgi apparatus to lymphocytes and macrophages.     

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.     

Biosynthesis and Immunolocalization of Lewis a-Containing N-Glycans in the Plant Cell

We recently demonstrated the presence of a new asparagine-linked complex glycan on plant glycoproteins that harbors the Lewis a (Lea), or Galbeta(1-3)[Fucalpha(1-4)]GlcNAc, epitope, which in mammalian cells plays an important role in cell-to-cell recognition. Here we show that the monoclonal antibody JIM 84, which is widely used as a Golgi marker in light and electron microscopy of plant cells, is specific for the Lea antigen. This antigen is present on glycoproteins of a number of flowering and non-flowering plants, but is less apparent in the Cruciferae, the family that includes Arabidopsis. Lea-containing oligosaccharides are found in the Golgi apparatus, and our immunocytochemical experiments suggest that it is synthesized in the trans-most part of the Golgi apparatus. Lea epitopes are abundantly present on extracellular glycoproteins, either soluble or membrane bound, but are never observed on vacuolar glycoproteins. Double-labeling experiments suggest that vacuolar glycoproteins do not bypass the late Golgi compartments where Lea is built, and that the absence of the Lea epitope from vacuolar glycoproteins is probably the result of its degradation by glycosidases en route to or after arrival in the vacuole.     

Microtubule nucleation at the cis‐side of the Golgi apparatus requires AKAP450 and GM130

We report that microtubule (MT) nucleation at the Golgi apparatus requires AKAP450, a centrosomal γ‐TuRC‐interacting protein that also forms a distinct network associated with the Golgi. Depletion of AKAP450 abolished MT nucleation at the Golgi, whereas depletion of the cis‐Golgi protein GM130 led to the disorganisation of AKAP450 network and impairment of MT nucleation. Brefeldin‐A treatment induced relocalisation of AKAP450 to ER exit sites and concomitant redistribution of MT nucleation capacity to the ER. AKAP450 specifically binds the cis‐side of the Golgi in an MT‐independent, GM130‐dependent manner. Short AKAP450‐dependent growing MTs are covered by CLASP2. Like for centrosome, dynein/dynactin complexes are necessary to anchor MTs growing from the Golgi. We further show that Golgi‐associated AKAP450 has a role in cell migration rather than in cell polarisation of the centrosome–Golgi apparatus. We propose that the recruitment of AKAP450 on the Golgi membranes through GM130 allows centrosome‐associated nucleating activity to extend to the Golgi, to control the assembly of subsets of MTs ensuring specific functions within the Golgi or for transporting specific cargos to the cell periphery.     

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans‐cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis‐Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.     

Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.     

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.     

Localization of Sed5, a putative vesicle targeting molecule, to the cis- Golgi network involves both its transmembrane and cytoplasmic domains

The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family. These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism by which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno- EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes. However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein. This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.     

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.     

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.     

Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein.

The vesicular stomatitis virus glycoprotein forms noncovalently linked trimers in the endoplasmic reticulum before being transported to the Golgi apparatus. The experiments reported here were designed to determine if the extracellular domain of the glycoprotein contains structural information sufficient to direct trimer formation. To accomplish this, we generated a construct encoding G protein with the normal transmembrane and anchor sequences replaced with the sequence encoding 53 C-terminal amino acids from the Thy-1.1 glycoprotein. We show here that these sequences were able to specify glycolipid addition to the truncated G protein, probably after cleavage of 31 amino acids derived from Thy-1.1. The glycolipid-anchored G protein formed trimers and was expressed on the cell surface in a form that could be cleaved by phosphoinositol-specific phospholipase C. However, the rate of transport was reduced, compared with that of wild-type G protein. A second form of the G protein was generated by deletion of only the transmembrane and cytoplasmic domains. This mutant protein also formed trimers with relatively high efficiency and was secreted slowly from cells.     

Recycling of proteins between the endoplasmic reticulum and Golgi complex.

Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined. Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards.     

Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein

As Rift Valley fever (RVF) virus, and probably all members of the family Bunyaviridae, matures in the Golgi apparatus, the targeting of the virus glycoproteins to the Golgi apparatus plays a pivotal role in the virus replication cycle. No consensus Golgi localization motif appears to be shared among the glycoproteins of these viruses. The viruses of the family Bunyaviridae synthesize their glycoproteins, G(N) and G(C), as a polyprotein. The Golgi localization signal of RVF virus has been shown to reside within the G(N) protein by use of a plasmid-based transient expression system to synthesize individual G(N) and G(C) proteins. While the distribution of individually expressed G(N) significantly overlaps with cellular Golgi proteins such as beta-COP and GS-28, G(C) expressed in the absence of G(N) localizes to the endoplasmic reticulum. Further analysis of expressed G(N) truncated proteins and green fluorescent protein/G(N) chimeric proteins demonstrated that the RVF virus Golgi localization signal mapped to a 48-amino-acid region of G(N) encompassing the 20-amino-acid transmembrane domain and the adjacent 28 amino acids of the cytosolic tail.