Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.     

Cholesterol loading induces a block in the exit of VSVG from the TGN

Recent work from our laboratory demonstrated that increased cellular cholesterol content affects the structure of the Golgi apparatus. We have now investigated the functional consequences of the cholesterol-induced vesiculation of the Golgi apparatus and the role of actin for these changes. The results showed that cholesterol-induced vesiculation and dispersion of the Golgi apparatus is a reversible process and that reversal can be inhibited by cytochalasin D, an actin-disrupting reagent. Furthermore, electron microscopy revealed that jasplakinolide, which stabilizes actin filaments, prevented the dispersion, but not the vesiculation of the Golgi cisternae. Importantly, the different Golgi markers seemed to be separated even after vesiculation. To investigate whether transport through the different steps of the exocytic pathway was affected in cholesterol-treated cells, we visualized ER to plasma membrane transport by using ts045-VSVG-GFP. In COS-1 cells expressing ts045-VSVG-GFP increased cholesterol levels did not affect transport of VSVG into the vesiculated Golgi apparatus. However, increased levels of cholesterol resulted in retention of the nascent G protein in vesicles with the TGN-marker TGN46. Biotinylation of cell surface molecules to quantify arrival of VSVG at the plasma membrane confirmed that cholesterol treatment inhibited export of the VSVG protein. In conclusion, the data show that transport of VSVG into/through a vesiculated Golgi is feasible, but that cholesterol loading inhibits exit of VSVG from the vesicles containing TGN markers. Furthermore, the data illustrate the importance of actin filaments for Golgi structure.     

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 μg/ml) for 1 h at 37 °C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a marked perinuclear fluorescence. In addition, MAM-6 fluorescence was observed in swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-μm secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

Targeting of membrane proteins to endosomes and lysosomes

The pathways involved in targeting membrane proteins to lysosomes are extraordinarily complex. Newly synthesized proteins in the ER are transported to the Golgi complex, and upon arrival at the trans Golgi network (TGN) are targeted either directly to endosomes, or first to the cell surface from where they can be rapidly internalized into the endocytic pathway for delivery to lysosomes. The routes to endosomes are specified by sorting motifs in the cytoplasmic tails of the proteins that are recognized at the TGN or plasma membrane. The molecular details of these processes are just emerging.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane-mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH(4)Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that approximately 30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.     

pH-independent retrograde targeting of glycolipids to the Golgi complex

A small fractionof the molecules internalized by endocytosis reaches the Golgi complexthrough a retrograde pathway that is poorly understood. In the presentwork, we used bacterial toxins to study the retrograde pathway in Verocells. The recombinant B subunit of verotoxin 1B (VT1B)was labeled with fluorescein to monitor its progresswithin the cell by confocal microscopy. This toxin, which bindsspecifically to the glycolipid globotriaosyl ceramide, enteredendosomes by both clathrin-dependent and -independent pathways,reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics ofinternalization and the subcellular distribution of VT1B were virtuallyidentical to those of another glycolipid-binding toxin, the B subunitof cholera toxin (CTB). Retrograde transport of VT1B and CTB wasunaffected by addition of weak bases in combination with concanamycin,a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that theseagents neutralized the luminal pH of the compartments where the toxinwas located. Therefore, the retrograde transport of glycolipids differsfrom that of proteins like furin and TGN38, which require an acidicluminal pH. Additional experiments indicated that the glycolipidreceptors of VT1B and CTB are internalized independently and not aspart of lipid "rafts" and that internalization is cytochalasininsensitive. We conclude that glycolipids utilize a unique,pH-independent retrograde pathway to reach compartments of thesecretory system and that assembly of F-actin is not required for thisprocess.

     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.     

Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.     

Lipid raft-independent B cell receptor-mediated antigen internalization and intracellular trafficking

The Ag-specific B cell receptor (BCR) expressed by B lymphocytes has two distinct functions upon interaction with cognate Ag: signal transduction (generation of intracellular second messenger molecules) and Ag internalization for subsequent processing and presentation. While it is known that plasma membrane domains, termed lipid rafts, are involved in BCR-mediated signal transduction, the precise role of plasma membrane lipid rafts in BCR-mediated Ag internalization and intracellular trafficking is presently unclear. Using a highly characterized model system, it was determined that while plasma membrane lipid rafts can be internalized by B lymphocytes, lipid rafts do not represent a major pathway for the rapid and efficient internalization of cell surface Ag-BCR complexes. Moreover, internalized plasma membrane lipid rafts are delivered to intracellular compartments distinct from those to which the bulk of internalized Ag-BCR complexes are delivered. These results demonstrate that B lymphocytes, like other cell types, possess at least two distinct endocytic pathways (i.e., clathrin-coated pits and plasma membrane lipid rafts) that deliver internalized ligands to distinct intracellular compartments. Furthermore, Ag-BCR complexes differentially access these two distinct internalization pathways.     

Delivery of lectin-labeled membrane to the trans-Golgi network and secretory granules in cultured atrial myocytes

To examine whether and how internalized plasma membrane components are routed to the compartment of the biosynthetic-exocytic pathway in cultured atrial myocytes, the plasma membrane labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was traced electron microscopically by cytochemical detection of HRP. The WGA-HRP label was internalized via a coated pit-small vesicle pathway and reached vacuoles and endosomes by 3 min. Labeled endosomes comprised vacuoles and tubular elements containing reaction product. By 15 min, similar tubular structures containing reaction product accumulated in the area of the trans-Golgi network (TGN). The labeled TGN consisted of interconnected tubular elements, which often connected to atrial granules containing reaction product. In contrast, neither native HRP nor Lucifer Yellow reached Golgi elements or atrial granules. These results suggest that a proportion of the plasma membrane labeled with WGA-HRP is delivered to endosomes, from which tubules might bud off to transfer the tracer molecules to the TGN, where the lectin conjugate and associated membranes are packaged into atrial granules.     

Demonstration of angiotensin II-induced Ras activation in the trans-Golgi network and endoplasmic reticulum using bioluminescence resonance energy transfer-based biosensors

Previous studies have demonstrated that molecules of the Ras signaling pathway are present in intracellular compartments, including early endosomes, the endoplasmic reticulum (ER), and the Golgi, and suggested that mitogens can regulate Ras activity in these endomembranes. In this study, we investigated the effect of angiotensin II (AngII) on intracellular Ras activity in living HEK293 cells expressing angiotensin type 1 receptors (AT(1)-Rs) using newly developed bioluminescence resonance energy transfer biosensors. To investigate the subcellular localization of AngII-induced Ras activation, we targeted our probes to various intracellular compartments, such as the trans-Golgi network (TGN), the ER, and early endosomes. Using these biosensors, we detected AngII-induced Ras activation in the TGN and ER, but not in early endosomes. In cells expressing a cytoplasmic tail deletion AT(1)-R mutant, the AngII-induced response was enhanced, suggesting that receptor internalization and β-arrestin binding are not required for AngII-induced Ras activation in endomembranes. Although we were able to demonstrate EGF-induced Ras activation in the plasma membrane and TGN, but not in other endomembranes, AG1478, an EGF receptor inhibitor, did not affect the AngII-induced response, suggesting that the latter is independent of EGF receptor transactivation. AngII was unable to stimulate Ras activity in the studied compartments in cells expressing a G protein coupling-deficient AT(1)-R mutant ((125)DRY(127) to (125)AAY(127)). These data suggest that AngII can stimulate Ras activity in the TGN and ER with a G protein-dependent mechanism, which does not require β-arrestin-mediated signaling, receptor internalization, and EGF receptor transactivation.     

Sorting of sphingolipids in the endocytic pathway of HT29 cells

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed. After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensin-induced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution. By contrast, C6-NBD-sphingomyelin does not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments. Also, in monensin-treated cells no change in the distribution of fluorescence was observed. Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids. In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin.     

Two lysosomal membrane proteins,LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.     

The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.     

The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.     

The dynamic nature of the Golgi complex

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.     

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.