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© The Rockefeller University Press,
0021-9525/1999//1457 $5.00
The Journal of Cell Biology, Volume 147, Number 7,
, 1999 1457-1472
Original Article |
ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro
ostermj{at}ctrvax.vanderbilt.edu
Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.
Key Words: Golgi apparatus • in vitro transport • secretion • transport vesicles • ER
© 1999 The Rockefeller University Press
THE intermediate compartment is the compartment through which secretory proteins pass before they reach the Golgi apparatus. It consists of vesicle clusters and tubular networks (or vesicular tubular clusters, VTCs; for review, see Bannykh and Balch 1997), and it represents a compartment in which long-lived resident proteins of the early secretory pathway are separated from the secretory flow and recycled to the ER (Pelham 1996). Assembly of the intermediate compartment is usually studied in the context of protein exit from the ER. The goal of this study is to shed light on the next step, transport between the intermediate compartment and the Golgi apparatus.
Important progress in our understanding of protein transport from the ER to the Golgi apparatus was made by visualizing transport in living cells. When a secretory protein is fused to green fluorescent protein, its transport can be directly observed by fluorescence microscopy (Presley et al. 1997; Scales et al. 1997). These studies demonstrated that ER-derived transport vesicles rapidly assemble into larger structures, the previously described VTCs, which form close to ER exit sites. These VTCs appear at the light microscopy level as punctate objects that travel along microtubules towards the Golgi apparatus. Proteins that must be retrieved to the ER are removed from VTCs in a coat protein I (COP I)-dependent process (Letourneur et al. 1994; Lewis and Pelham 1996). After VTCs have reached the Golgi apparatus, they appear to merge with it. However, visualization of protein transport with green fluorescent protein has done little to elucidate the role of transport vesicles in transport to and through the Golgi apparatus.
Different models have been proposed to explain protein transport through the Golgi apparatus (Farquhar and Palade 1998). Secretory cargo passes through the Golgi apparatus from cis to trans. Transport in the retrograde direction retrieves Golgi-localized proteins and separates them from the secretory flow. The vesicular transport hypothesis predicts that transport between neighboring compartments occurs by vesicular transport in both directions (Farquhar 1985; Orci et al. 1997). The cisternal maturation hypothesis proposes that the cisternae of the Golgi stack are anterograde transport intermediates that are in different stages of maturation (Beams and Kessel 1968; Becker and Melkonian 1996; Bonfanti et al. 1999). An important prediction of the cisternal maturation hypothesis is that Golgi cisternae form constantly anew on the cis-side of the organelle, possibly by fusion of ER-derived transport intermediates with Golgi-derived transport vesicles. The retrograde transport of Golgi enzymes between cisternae in the trans-to-cis direction could drive the maturation of cisternae while maintaining a stationary enzyme distribution across the stack. It has been proposed that the competition of different Golgi enzymes for retrograde transport could explain the observed distribution of enzymes in the Golgi stack (Glick et al. 1997).
A less well-developed alternative to these two currently prevailing models is that the cisternae of Golgi apparatus are continuous. Such a continuity would require that the anterograde flow of secretory cargo is coupled to a separate retrograde transport of resident Golgi enzymes. The interactions of Golgi enzymes with their substrates, as well as interactions between the Golgi enzymes themselves, might explain why not all Golgi enzymes are evenly distributed over the Golgi stack (Lippincott-Schwartz et al. 1998). While cisternae often appear to be distinct from each other when observed by electron microscopy, transient connections might rapidly form and break. They could be highly unstable and would be difficult to observe in fixed specimens; nevertheless, there are well-documented examples of continuity between Golgi cisternae (Rambourg et al. 1993; Clermont et al. 1994).
Together with morphology and genetics, in vitro transport assays are used to study intra-Golgi protein transport. Rothman and coworkers characterized an in vitro complementation assay in which Golgi membranes are isolated from wild-type (wt) cells and from vesicular stomatitis virus (VSV)-infected glycosylation-defective mutant cells (Fries and Rothman 1980). When these membranes are incubated together under the appropriate conditions, VSV-glycoprotein (VSV-G) that is enclosed in mutant cell membranes is glycosylated by Golgi enzymes enclosed in the wt membranes. A morphological analysis of a related assay had provided evidence that this assay might reconstitute the anterograde transport of secretory cargo within the Golgi stack (Braell et al. 1984). It was further suggested that such anterograde transport occurs in COP I–coated vesicles that form on Golgi membranes when incubated in vitro (Orci et al. 1986). Indeed, it was shown that VSV-G could be transferred in this system in a COP I–dependent reaction (Ostermann et al. 1993).
However, the difficulty of interpreting the many complex and sometimes contradictory results from in vitro experiments has resulted in criticism of this approach, validating the assay only as a tool to identify components (Mellman and Simons 1992). Some of the results are not readily reconciled with the anterograde vesicular transport model that had originally been applied to interpret the experiments. For example, Golgi enzymes are not excluded from COP I vesicles (Ostermann et al. 1993; Sönnichsen et al. 1996; Lanoix et al. 1999), and indeed, transport of Golgi enzymes in the retrograde direction may be sufficient to account quantitatively for the glycosylation that is measured in this in vitro assay (Love et al. 1998). The situation is further complicated by the possibility that in vitro complementation assays might measure a combination of several different reactions. Direct fusion of Golgi membranes, transport through continuities of Golgi subcompartments, and vesicular transport in both the anterograde and retrograde direction would all contribute to glycosylation of VSV-G by wt Golgi enzymes in this assay.
That transport occurs even when COP I assembly was inhibited has questioned even the relevance of the COP I pathway in Golgi transport (Orci et al. 1991; Elazar et al. 1994; Taylor et al. 1994; Happe and Weidman 1998; Dominguez et al. 1999). However, we recently discovered that cellular homogenates contain small membranous structures that are highly active in the complementation assay, and we found these membranes even when cells were homogenized under gentle conditions (Love et al. 1998). They were of much smaller size than the bulk of the Golgi membranes in homogenates. Relative to the amount of Golgi enzyme activity contained in these membranes, they were found to be much more active than routinely prepared Golgi membranes. At that time, we proposed the hypothesis that these active membranes are intermediates in the retrograde transport of Golgi enzymes, possibly generated in a COP I–dependent reaction in cells before homogenization, and possibly identical with the abundant vesicles and small pleiomorphic structures seen in EM reconstitution of the Golgi apparatus (Ladinsky et al. 1999).
Two goals were pursued in the study that is summarized here. First, we assessed critically the target compartment in which the VSV-G protein must reside to become glycosylated with the highest efficiency. We found that glycosylation occurred with the highest yield not when VSV-G resided in the Golgi apparatus or in the ER but when it was arrested in transit between them. Second, we determined what type of transport was reconstituted when transport was studied after removal of preexisting transport vesicles. We found that Golgi enzymes gained access to VSV-G not by direct fusion of ER/Golgi intermediates with each other or the Golgi stack, but by COP I–dependent transport.
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Materials and Methods |
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supplemented with 10% fetal calf serum plus penicillin and streptomycin in a 5% CO2 atmosphere at 37°C and split 1 to 10 every 2 d. For large scale production of cells for isolation of membrane fractions or cytosol, cells were grown in spinner flasks until confluent.
VSV Infection
15B cells from a confluent spinner culture were pelleted, resuspended in 50 ml of infection media (serum free media, 1 ml VSV, 0.1 mg/ml actinomycin D, 25 mM Hepes, pH 7.2), and incubated for 45' at 37°C in a small spinner flask. 200 ml of media with serum were added, and the infection was continued for an additional 2 h 15 min.
Pulse–Chase Labeling
Cells were pelleted by centrifugation, washed once, and resuspended in 10 ml of methionine-free media per 1 ml of cell pellet. After 5 min at 37°C, 0.1 to 0.5 mCi per ml of 35S-methionine/cysteine were added, and cells were incubated for an additional 5 min at 37°C (pulse). The labeling mixture was then diluted into a spinner flask containing a large volume of methionine-containing media in a water bath set to the desired temperature to be incubated for the indicated times (chase). For an additional chase at 37°C, aliquots were withdrawn and incubated for the indicated times in a water bath at 37°C.
Gradient Fractionation of Cellular Homogenates
Cells were collected, washed once in PBS, and resuspended in 4 ml per ml cell pellet of ST buffer (200 mM sucrose, 10 mM Tris, pH 7.2). This suspension was homogenized in a ball bearing homogenizer. For fractionation by sucrose density gradient centrifugation, homogenate was loaded on a 5-ml linear sucrose gradient (20–50% weight per weight) in KHM (150 mM KCl, 10 mM Hepes, pH 7.2, 2.5 mM MgOAc) buffer. Gradients were centrifuged for 16 h in an SW55 rotor at 50,000 rpm at 4°C. 10 fractions were collected from the top. Velocity sedimentation was done by loading homogenate on a 15 to 35% sucrose/KHM gradient and centrifuging it in an SW55 centrifuge tube for 20 min at 50,000 rpm. 10 fractions were collected from the top.
Preparation of Cisternal wt Golgi Membranes
To prepare Golgi-enriched membranes, homogenate was centrifuged for 5 min at 1,000 g. The supernatant was loaded on a 1-ml cushion of 40% sucrose in 10 mM Tris, pH 7.2, and Golgi membranes were pelleted on this cushion by a 10-min centrifugation at 17,500 g. The bottom 2 ml were collected, mixed in an SW40 tube with 62% sucrose to 
40% final, and overlaid with 4 ml of 35% and 3 ml of 29% sucrose. After 90 min centrifugation at 40,000 rpm, Golgi membranes were harvested at the interface between 29% and 35% sucrose. For small scale preparations of Golgi membranes from cells after pulse–chase incubations, homogenates were mixed with 62% sucrose to 37.5% sucrose final and were overlaid with 2 ml 35% and 1 ml 29% sucrose in an SW55 centrifuge tube. After 90 min centrifugation at 50,000 rpm, Golgi membranes were harvested at the interface between 29% and 35% sucrose. To prepare salt-washed Golgi membranes, Golgi membranes harvested after flotation were diluted with 4 vol of 10 mM Tris, pH 7.2, and KCl was added to 250 mM final concentration. After 20 min incubation on ice, the mixture was loaded on 1 ml 40% and 3 ml 20% sucrose in an SW40 centrifuge tube. Golgi membranes were pelleted through the 20% layer on the 40% cushion by 20 min centrifugation at 15,000 rpm and were harvested at this interface.
Isolation of Membranes from Supernatant of Permeabilized Cells
To prepare fractions enriched in active transport intermediates, cells resuspended in ST were frozen in liquid nitrogen and kept at –80°C until use. Cells were thawed in a water bath at room temperature. Permeabilized cells and other debris were removed by centrifugation at 1,000 g and 17,500 g. In some experiments, vesicles were separated from cytosol by loading 400 µl of the supernatant on 400 µl 20% sucrose and 200 µl 40% sucrose. After 15 min centrifugation at 100,000 rpm in the TLA120.2 rotor, the top 600 µl was discarded and the bottom 400 µl was collected. For large scale preparations, membranes in the supernatant of permeabilized cells were pelleted by centrifugation for 20 min at 75,000 rpm in the TLA100.4 rotor, resuspended in KHM buffer and fractionated by velocity sedimentation on a 30-ml linear 15–35% sucrose/KHM gradient in a SW28 tube (25' at 28,000 rpm). Vesicle containing fractions were identified by measuring GalT activity and were pooled and layered on top of a 0.75-ml cushion of 50% iodixanol in HM. Vesicles were pelleted on this cushion by 3 h centrifugation at 40,000 rpm in a SW40 rotor. 1.5 ml were collected from the bottom and mixed with 0.5 ml of 50% iodixanol in HM. A step gradient of 2 ml of 25% and 1 ml of 10% iodixanol in KHM was layered on top of this sample, and the gradient was centrifuged for 3 h at 55,000 rpm in a SW55 rotor. Vesicles were harvested at the interface between the 10 and 25% layers.
Electron Microscopy
Membrane fractions were prepared for routine electron microscopy using a filtration apparatus for random sampling exactly as described previously by Dominguez et al. 1999. Membranes associated with magnetic beads were processed identically to the conditions described in Dominguez et al. 1999, except that the material was retained by magnets during throughout the washing and fixation procedures.
Preparation of Cytosol
wt or 15B cells were gently broken by a cycle of freezing and thawing as described above or were homogenized in a ball bearing homogenizer. Cellular debris and membranes were pelleted by subsequent centrifugations at 1,000, 17,500, and 250,000 g. Repeated centrifugations at 250,000 g were done when active vesicles needed to be completely removed from wt cytosol. To remove low molecular mass components in cytosol, it was either passed over a PD10 gel filtration column, or it was dialyzed against 25 mM Tris, pH 7.2, 50 mM KCl using dialysis tubing with a molecular weight cutoff of 12–14 kD. To deplete coatomer from cytosol, 100 µl protein A–Sepharose slurry was incubated with 100 µl CM1A10 ascites fluid. The Sepharose beads were reisolated and washed. Half of the beads were incubated with 150 µl cytosol for 20 min at 4°C. Cytosol was recovered and incubated with the other half of the beads for 60 min at 4°C.
Immunoisolation of Membranes
M450 (for gel electrophoresis and enzyme assays) or M500 (for electron micrographs) magnetic beads were cross-linked with anti-rabbit IgG according to the manufacturer's instructions. Typically, 10 and 40 µl of the bead suspension was used per immunoisolation. Beads were preincubated with affinity-purified p23 cytoplasmic tail antibodies and reisolated with a magnet. Antibody coated magnetic beads were incubated with membranes in immunoisolation buffer (KHM buffer plus 0.2 M sucrose and 0.5 mg/ml milk powder) for 2 h in the cold room with gentle agitation. Beads were reisolated with a magnet and washed repeatedly in buffer (last wash without milk powder). Beads were either extracted with electrophoresis buffer (for gel electrophoresis and Western blotting) or in NAGT, GalT, or mannosidase assay buffers. Membranes that remained in the supernatant after immunoisolation were pelleted by ultracentrifugation. For EM analysis, samples were frozen for shipment and storage.
Enzyme Assays
Mannosidase activity was measured in 100-µl samples. Each contained 58 µl 0.2 M Na-phosphate, pH 6.0, 20 µl 20 mM 4-methylumbelliferyl--D mannopyranoside in DMSO, 2 µl 10% Triton X-100, and 20 µl sample (or water). After 60 min at 37°C, samples were diluted in 1 ml 0.25 M sodium carbonate. Fluorescence was measured at 448 nm for emission and 364 nm for excitation light. NAGT activity was measured in 100 µl samples. Each contained 5 µl 1 M Tris, pH 6.8, 0.33 µl 3 M KCl, 1 µl 1 M MnCl2, 1 µl 1 M MgCl2, 10 µl 20 mg/ml ovalbumin, 1 µl 10% Triton X-100, 1 µl 3H-UDP-GlcNAc (0.1 mCi/ml), and 20 µl sample (or water). After 2 h at 37°C, 1 ml of ice-cold 1% phosphotungstic acid in 0.5 M HCl was added to each sample. After 10 min on ice, the precipitate was pelleted by a short centrifugation (5'' in a microcentrifuge). The pellet was resuspended in 1 ml of phosphotungstic acid in 0.5 M HCl, centrifuged again, resuspended in 1 ml ice-cold 95% ethanol, and pelleted again. Pellets were completely dissolved in 200 µl of 1% SDS and 50 µl of 2 M Tris base and mixed with 15 ml scintillation cocktail to determine the amount of 3H bound to protein. GalT activity was measured in 50-µl samples. Each contained 2.5 µl 1 M Tris, pH 6.8, 1 µl 10% Triton X-100, 2 µl 1 M MnCl2, 5 µl 10 mg/ml ovalbumin, 2.5 µl 40 mM ATP, 1 µl 3H-UDP-Gal (0.1 mCi/ml), and 10 µl sample (or water). After 1 h at 37°C, samples were spotted in the middle of 2.5-cm-wide squares drawn on filter paper. Filters were incubated in 10% trichloroacetic acid for 10 min at room temperature, rinsed quickly with water and washed three times for 5 min with water. Filters were dried, each square was cut out, and filter-bound radioactivity was measured by scintillation counting.
In Vitro Complementation Assay
Unless indicated otherwise, 50 µl incubation would include 5 µl wt Golgi membranes, 5 µl 15B Golgi membranes isolated from VSV-infected cells, 15 µl CHO cytosol, 75 mM ATP, 3 mM creatine phosphate, and 12 IU/ml creatine kinase. The final buffer conditions contained 25 mM Hepes, pH 7.2, 2.5 mM MgOAc, 50 mM KCl, and 200 mM sucrose. When salt-washed Golgi membranes were used, 200 ng/ml N-ethylmaleimide sensitive factor was also added. Incubations were done for 1 h at 37°C. At the end of the incubation, Golgi membranes were pelleted by centrifugation (10 min 17,500 g), resuspended in 20 µl 50 mM citrate, pH 5.5, 1 mM DTT, and 0.4% SDS, and denatured for 3 min at 95°C. 20 µl of 50 mM citrate were added, and 1.7 mIU endo H was added when indicated. After at least 1 h incubation at 37°C, an aliquot of the digest was mixed with electrophoresis sample buffer and separated by gel electrophoresis. To observe late Golgi modifications, 4 mM each of UDP-galactose and CMP-sialic acid were added during the initial incubation. When indicated, 0.2 IU/ml of neuraminidase was added together with endo H.
Determination of BFA Sensitivity
To assure that cytosol would not contain any wt Golgi-derived membranes, cytosol prepared from 15B cells was used for most BFA inhibition experiments. A 10 mg/ml solution of BFA (purchased from Calbiochem) in ethanol was prepared and kept at –20°C until use. To add BFA to the transport assay, the BFA stock solution was diluted 1 to 100 in water, and 10 µl of this dilution was included in each incubation mixture.
Gel Electrophoresis, Phosphorimaging, and Western Blotting
Proteins were separated by denaturing protein gel electrophoresis using a Biorad Miniprotean® 2 or 3 gel system. For optimal resolution of the differently glycosylated forms of VSV-G, proteins were separated on a 3.5-ml 10% acrylamide gel (acrylamide to bisacrylamide, 37.5:1) with a 1-ml 5% stacking gel. After 85 min electrophoresis at 200 V, gels were fixed in 40% methanol and 10% acetic acid and dried on filter paper. Visualization of radioactive VSV-G was done by phosphorimaging. Typically, exposures were chosen with a maximum intensity (100% black) between 102 and 103. The minimum intensity (0% black) was set to 1/100 of the maximum, and intensities in between were linearly assigned gray scale values from 0 to 100%. For detection of p25 by Western blotting, the acrylamide concentration was increased to 14%, and electrophoresis was done for 40 min. Proteins were electrophoretically transferred on PVDF filter, and the filter was incubated with a polyclonal antiserum raised against p25 (Dominguez et al. 1998). Bound antibody was detected by chemiluminescence.
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Accumulation of Secretory Cargo at 15 and 20°C
In vitro complementation assays to measure intra-Golgi transport are most frequently performed by measuring incorporation of radiolabeled sugar into VSV-G. As we had done previously (Love et al. 1998), we used this assay in a modified form by directly labeling the VSV-G protein in the glycosylation-deficient cells. To accumulate radiolabeled VSV-G in different parts of the secretory pathway, we labeled newly synthesized VSV-G (pulse) and then incubated cells at different temperatures for different times to allow forward transport of VSV-G (chase). When cells are incubated at 15°C, secretory cargo leaves the ER, but instead of being transported to the Golgi apparatus it accumulates in ER/Golgi intermediates that remain in the cellular periphery (Saraste and Kuismanen 1984). When cells are incubated at 20°C, secretory proteins accumulate in late Golgi compartments (Matlin and Simons 1983). Golgi processing of VSV-G was detected by measuring its sensitivity to endoglycosidase H (endo H) that distinguishes between the ER and Golgi forms of N-linked glycans. As expected, at 20°C VSV-G was slowly converted from an endo H–sensitive to an endo H–resistant form (Fig. 1 A). When cells were incubated for up to 3 h at 15°C, VSV-G did not become endo H resistant. When these cells were subsequently incubated at 37°C, glycosylation resumed, and after 20 min VSV-G had mostly been converted to the endo H–resistant form (Fig. 1 B). This gives us a lower limit for the time VSV-G resides in Golgi membranes, but it is very likely that VSV-G remains in the stack for longer than these kinetics suggest. VSV-G transport can be more directly followed by fluorescence microscopy, and in such experiments VSV-G can still be found in the Golgi apparatus for as long as 1 h after release of the 15°C block (for example, see Rojo et al. 1997). Furthermore, Golgi enzyme distributions overlap, and even late-acting enzymes can be found in early Golgi compartments (Nilsson et al. 1993; Velasco et al. 1993; Lovelock and Lucocq 1998).
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Sucrose Gradient Fractionation of ER/Golgi Intermediates
We studied the fractionation of VSV-G–containing membranes after the 15°C chase by velocity sedimentation and density gradient centrifugation to confirm that the bulk of VSV-G had exited the ER during the 15°C chase. After pulse labeling of newly synthesized VSV-G, half of the cells were kept on ice, and the other half was incubated in the presence of an excess of unlabeled amino acids for 3 h at 15°C. We prepared homogenates from both and fractionated them by sucrose gradient centrifugation. After equilibrium density gradient centrifugation, we found that approximately half of the radiolabeled VSV-G in membranes of cells that had been incubated at 15°C was redistributed to a lighter membrane fraction (Fig. 2 A, fractions 4–6). When the 15°C chase was omitted, much of the VSV-G protein was found at higher densities in fractions 7–10.
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In summary, at 15°C VSV-G redistributed into lighter membranes that were larger and less heterogeneous in size, and we conclude that the bulk of VSV-G had exited the ER during the 15°C chase.
Immunoisolation of ER/Golgi Intermediates with p23 Antibodies
Our results (Fig. 1 C), as well as earlier results (Balch et al. 1986), indicated that Mann I processing is only partially inhibited at 15°C, and this suggests that VSV-G had entered the Golgi apparatus. Equally possible is that a fraction of Mann I might be present in ER/Golgi intermediates.
In intact cells, ER/Golgi intermediates are readily distinguishable from Golgi membranes due to their peripheral localization, but they are not readily separable from Golgi membranes in cellular homogenates. A clear separation of the two may well be impossible. However, ER/Golgi intermediates are enriched in proteins of the p24 family (Schimmoller et al. 1995; Stamnes et al. 1995; Belden and Barlowe 1996; Rojo et al. 1997; Dominguez et al. 1998). We determined whether it was possible to make use of an antibody that recognized the cytoplasmic tail of the p23 protein, one member of this family, to distinguish between membranes of the Golgi stack on the one hand and ER/Golgi intermediates and the cis-Golgi network on the other (Rojo et al. 1997). As p24 proteins could be present in smaller amounts in the Golgi stack (Dominguez et al. 1998), we did not expect that immunoisolation with p23 antibodies would allow us to obtain a true separation of ER/Golgi intermediates and Golgi membranes. However, we did hope to find a depletion of Golgi enzymes from the immunoisolated membranes as Golgi enzymes are most concentrated in the membranes of the stack where the concentration of p24 proteins is the lowest.
Electron microscopy allowed us to visualize the membranes that are immunoisolated with the p23 antibody. We coated magnetic beads with anti-rabbit IgG antibody together with or without affinity-purified p23 antibody and incubated these with Golgi-enriched membranes. After incubation, we recovered the beads with a magnet and washed them to remove any material that was not tightly bound. The beads were then processed for electron microscopy (Fig. 3). In the absence of this antibody, no membranes were found bound to the beads. However, when p23 antibodies were present, beads were coated with membranes that were enriched in the anastomosing sectioned tubules characteristic of ER/Golgi intermediates and the cis-Golgi network (Dominguez et al. 1998). However, in addition to these, we also observed in some images clear evidence for cisternal membranes that appeared to be derived from the Golgi stack in continuity with these cis-Golgi tubules. The variability in recovery of stacked Golgi membranes and the abundance of tubular elements in these images suggests that, as we had expected, the p23 antibody allows us to prepare a membrane fraction that is enriched in cis-Golgi and pre-Golgi intermediates, but also contains a variable amount of associated Golgi membranes.
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In Vitro Glycosylation of VSV-G in ER/Golgi Intermediates
We employed the pulse–chase conditions described above to introduce labeled VSV-G into the ER, ER/Golgi intermediates, or the Golgi stack. This allows us to determine where in the secretory pathway VSV-G must reside to be efficiently glycosylated by in vitro complementation. When we incubated 15B membranes containing labeled VSV-G in the 15°C compartment with wt Golgi membranes, we observed that VSV-G was converted to the endo H–resistant form with very high efficiency (Fig. 5 A). As a control, wt membranes were omitted, and under those conditions VSV-G remained endo H sensitive. VSV-G also remained endo H sensitive when incubations were done on ice, or without addition of cytosol, ATP, or the activated sugar UDP-GlcNAc (data not shown). We carefully quantified the glycosylation yield in this reaction to assure that we could reproducibly obtain high glycosylation yields in vitro after accumulation of VSV-G in the 15°C compartment (Table ). We added saturating amounts of wt membranes so that we could be sure that glycosylation of VSV-G had reached the maximal level that could be obtained under these conditions. In the series of experiments that were quantified for this table, we used six different batches of membranes and tested them under experimental conditions that were typical for the experiments shown here. We found that 68 ± 6% of VSV-G became endo H resistant at the end of the incubation indicating reproducibility in comparing different batches of membranes.
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To determine whether the in vitro complementation might be the result of fusion of ER/Golgi intermediates containing newly synthesized Golgi enzymes in transit from the ER to the Golgi, we isolated membranes from wt cells that had been treated with cycloheximide. We found that these were equally active in the complementation assay as the membranes isolated from control cells (Fig. 5 C). We also tested how the yield of the complementation reaction changed when we either omitted the 15°C chase, shortened the time of the 15°C chase, or increased the chase temperature to 17.5 or 20°C (Fig. 5 C). We found that the yield of the complementation reaction dropped when the chase was done for the same time but at an even slightly higher temperature. When the chase time at 15°C was shortened to 1.5 h instead of 3 h, less of the total VSV-G was recovered in the Golgi-enriched fraction, but the relative yield of the complementation reaction was unaffected. Even when the chase after pulse labeling was entirely omitted, a fraction of VSV-G became endo H resistant. This could indicate transport of Golgi enzymes to the ER, as has been suggested by some, but the evidence remains contradictory (Cole et al. 1998; Shima et al. 1998; Storrie et al. 1998). More likely is that a small fraction of VSV-G exited the ER during the labeling step, and this fraction of VSV-G is enriched during preparations of membranes used in the in vitro transport assay (see Fig. 2 A; fractions 4–6 are the membranes used in the in vitro assay).
In summary, we conclude that in vitro complementation becomes efficient as VSV-G exits the ER and reaches ER/Golgi intermediates. As VSV-G progresses into the Golgi stack, in vitro complementation again becomes inefficient. We conclude that ER/Golgi intermediates are the optimal target compartment for the in vitro transport of wt Golgi enzymes.
Completion of VSV-G Glycosylation in ER/Golgi Intermediates
The in vitro complementation assay that we used in our experiments relies on processing by NAGT I, and this is followed by Mann II processing so that VSV-G will become resistant to endo H. We were interested in determining whether later Golgi modifications could also occur in vitro. This would suggest that ER/Golgi intermediates contained at least small amounts of the enzymes that are required to complete Golgi processing of N-linked glycans. Such late-acting Golgi enzymes might be present in ER/Golgi intermediates of 15B cells, or they might be acquired by retrograde transport during the in vitro incubation. Indeed, the observation that antibodies against p23, a protein highly enriched in ER/Golgi intermediates and the cis-Golgi network, immunoisolated a significant fraction of GalT activity suggests physical continuity among these compartments.
We determined the time course of glycosylation after complementation of the 15B defect. In previous experiments, we had included UDP-N-acetyl glucosamine (UDP-GlcNAc), the activated sugar that is transferred by NAGT. Now, we also added UDP-galactose and CMP-sialic acid, the substrates for GalT and sialyl transferase, respectively (Fig. 6 A). We found that at intermediate time points during the incubation, a form of VSV-G appeared that was resistant to endo H and migrated with the same mobility as the endo H–resistant VSV-G that was seen when only UDP-GlcNAc was added (Fig. 6 A, endo H resistant). After longer time points, the mobility of endo H–resistant VSV-G decreased further, indicating the addition of more sugar molecules. After 2 h, most endo H–resistant VSV-G no longer migrated as a sharp band but was spread out over a wider area (Fig. 6 A, neuraminidase sensitive). To confirm that this increase in mobility was caused by addition of sialic acid, we digested VSV-G with neuraminidase. We found that after neuraminidase treatment, VSV-G migrated as a sharp band that was very similar in mobility to the transiently appearing endo H–resistant band.
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ER/Golgi Intermediates and Golgi Membranes Remain Distinct during Incubation
Having established in which compartment VSV-G must reside to be glycosylated by in vitro complementation, our attention turned towards studying the mechanism of this process. Specifically, we wished to determine whether in vitro complementation occurred as a consequence of dissociative vesicular transport, or by direct fusion of Golgi compartments.
So far, we provided evidence that VSV-G must reside in a pre-Golgi compartment at the beginning of the incubation. As VSV-G was still recovered with p23 antibodies at the end of the incubation, most of it was not separated from proteins of ER/Golgi intermediates. However, as membrane fusion occurs in this assay, ER/Golgi intermediates might directly fuse with wt Golgi membranes (Happe and Weidman 1998). If this had occurred, then VSV-G might still colocalize with p23, but the compartmental distinction between ER/Golgi intermediates and membranes of the Golgi stack might have been lost. We therefore determined whether the immunoisolation yield of Golgi marker enzymes and VSV-G changed after the in vitro incubation (Fig. 7). As expected, we found only a slight increase in the amount of Golgi enzyme activity that was recovered after immunoisolation, suggesting transport of Golgi enzymes to ER/Golgi intermediates. We also observed a similarly small decrease of VSV-G recovery, which may be caused by a separation of forward moving VSV-G from retrograde moving p23.
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Characterization of the wt Golgi Activity
Two results now suggest that cisternal Golgi membranes do not fuse with each other in this assay. First, complementation efficiency is maximal after the 15°C incubation and decreases thereafter. If all Golgi membranes were capable of fusion, then complementation should remain constant for as long as VSV-G resides in Golgi membranes. Second, as discussed in the preceding paragraph, we observed only small changes in the p23 immunoisolation after incubation.
We showed in past work that wt Golgi membranes can form functional Golgi enzyme-containing transport vesicles, and we showed that enough of them are formed to account for the observed glycosylation yield (Love et al. 1998). In a recently completed study, cargo selection into COP I vesicles was studied, and it was found that NAGT I is efficiently packaged into functional transport vesicles (Lanoix et al. 1999). However, an important and seemingly contradictory observation is that BFA, an inhibitor of COP I assembly, had no effect on transport (Orci et al. 1991; Elazar et al. 1994; Taylor et al. 1994; Happe and Weidman 1998; Dominguez et al. 1999).
We hypothesized that the BFA-resistant transport described earlier is fusion of preexisting transport intermediates. To determine whether this hypothesis was reasonable, we first quantified the activity of different sources of wt Golgi activity in this assay. The most active wt Golgi fraction that we could obtain were the small vesicles that remained in the supernatant of permeabilized cells (Love et al. 1998). We therefore prepared such a supernatant and compared its activity with the activity of large, cisternal wt Golgi membranes that were isolated in parallel (Fig. 8 A). To assure that transport was measured under identical conditions, the reaction in which cisternal Golgi membranes were used was supplemented with a supernatant fraction from which all vesicles were removed by ultracentrifugation. This assured that both incubations contained equal amounts of cytosol so that any differences detected between the different fractions were due to differences in activity of the membranes, rather than cytosolic factors. We found that based on volume, both the vesicle-containing supernatant fraction and the cisternal Golgi membrane fraction contained approximately equal activity. However, when we measured the amount of NAGT activity in each fraction, we found that the Golgi fraction contained 1 to 2 orders of magnitude more NAGT activity (Fig. 8 B).
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Transport in the Absence of Preexisting Transport Intermediates Is Inhibited by BFA
Before we could detect transport that originated from cisternal Golgi membranes, it was necessary to add a salt wash step to the isolation procedure to remove loosely bound transport intermediates. To confirm that this was necessary, we incubated Golgi membranes with 250 mM KCl and then pelleted the Golgi membranes by a medium speed centrifugation. Even though the bulk of the Golgi membranes were pelleted, the supernatant was approximately as active when incubated with 15B Golgi membranes as the total membranes from which the supernatant was derived (Fig. 10). No active membranes remained in the supernatant when salt was omitted. From this we conclude that active transport intermediates remain bound to Golgi membranes during their isolation. Unless removed, they are likely to be an important if not exclusive contributor to the assay signal.
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We found that when salt-washed wt-Golgi membranes were used in the in vitro complementation assay, the reaction was efficiently inhibited by 20 µg/ml BFA (Fig. 11 A). While lower concentrations are sufficient for the in vivo effects of BFA, the concentration used here is identical to the one used previously to inhibit COP I assembly in vitro (Donaldson et al. 1991). We also observed that the transport reaction with salt washed membranes was very sensitive to addition of ethanol, a known inhibitor of vesicle formation (Pfanner et al. 1989; Ktistakis et al. 1996). BFA was added as a solution in ethanol, but at the low concentration used here, the solvent had only a small inhibitory effect (Fig. 11 A, compare lanes 3 and 6 or 5 and 8).
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As BFA is not inhibitory but stimulatory when vesicles are added, it is established that BFA inhibition in the transport assay with salt-washed wt Golgi membranes is not caused by an inhibition of the fusion reaction. It also demonstrates that, as is predicted by our hypothesis of vesicular transport from wt to 15B Golgi, BFA inhibits by acting on wt membranes, and not on the 15B membranes. If, for example, BFA inhibited by preventing maturation of the 15B membranes, then we should observe this inhibition irrespective of the source of wt Golgi activity.
Transport Is Inhibited by Antibodies against Coatomer and Coatomer Depletion
The BFA inhibition suggests that COP I assembly is required for transport in this system, and a direct test of this hypothesis is to deplete cytosol of COP I components and show that it is inactivated. We found that even though cytosol was not completely inactivated by immunodepletion, its activity was significantly reduced (Fig. 12 A). We also determined whether addition of purified CM1A10 antibody inhibited transport. We found that as little as 5 ng antibody was partially inhibitory, and maximal inhibition was observed with 25 ng (Fig. 12 B). When wt activity was added as vesicles instead of cisternal membranes, no antibody inhibition was observed at any of the concentrations tested (Fig. 12 C). This allows us to conclude that just as BFA, the coatomer antibody inhibits transport at the stage of coat assembly on wt membranes. Membrane fusion with and glycosylation in 15B membranes are not inhibited.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Only one membrane fusion reaction appears to occur in vitro under our experimental conditions: the fusion of Golgi enzyme-containing transport vesicles with ER/Golgi intermediates. Two sources for Golgi enzyme-containing transport intermediates have been identified: preexisting transport intermediates, some of which are released when cells are permeabilized or when Golgi membranes are incubated with salt, and newly generated transport intermediates that are formed by Golgi membranes in a BFA-sensitive reaction. In earlier work, we had established that Golgi enzymes could be transported in COP I vesicles (Love et al. 1998; Lanoix et al. 1999). However, only with the reconstitution of a BFA-sensitive mode of transport have we eliminated the possibility that cisternal Golgi compartments might also fuse directly with each other, or that pre-Golgi intermediates might fuse directly with the Golgi stack (Happe and Weidman 1998).
The impact of this study on our understanding of the Golgi apparatus is straightforward. That ER/Golgi intermediates acquire Golgi enzymes is an important prediction of the cisternal maturation model, and it has now been confirmed. Future work can now focus on the mechanism by which the contents of ER/Golgi intermediates is exchanged during their conversion from an ER-derived to a Golgi-derived structure. This will provide important insights that are likely to be also applicable to other transport steps, such as transport through the Golgi stack or endocytosis. However, other than this promise, little has been accomplished here to understand the next transport step, progression through the Golgi stack. We propose that this transport is not reconstituted in our system. Possibly, transport through the Golgi stack requires components or specific conditions that have yet to be identified. For example, it may require tethering of vesicles to cisternae (Orci et al. 1998; Sönnichsen et al. 1998), or it may involve other modes of transport that are impossible to detect by an in vitro complementation assay that can only detect freely diffusible transport intermediates.
Additional work is needed to understand the nature of the active intermediates. At this time, we can say little more of them other than that they exist, that they are highly active, and that they must be removed if one wants to study a full round of transport. An unexpectedly large fraction of Golgi enzymes were found to cofractionate with these structures, and preliminary data from our group suggest a functional and structural heterogeneity in this membrane fraction. The activity described here, fusion with ER/Golgi intermediates, is likely to represent only one of several intermediates present in this fraction. Further morphological and biochemical characterization of these membranes is likely to provide insights into the mechanism of later transport steps. The exceptionally high activity of these membranes is the principal reason why we expect them to be of significance, rather than an artifact of cell breakage. However, the validity of this study's conclusions is not put in question by this uncertainty. Even if the membranes we think of as retrograde transport intermediates were caused by an artifact, they would still have to be removed before transport between Golgi membranes could be studied.
The in vitro assay used here has a long history of controversy. However, only two findings, both entirely plausible and readily observable by independent approaches, were required to resolve this puzzle. The first is that COP I vesicles contain Golgi enzymes, and, due to the catalytic effect, this means that transport of the enzyme will dominate the assay signal. The second finding is that cellular homogenates are produced from growing and secreting cells and contain steady state levels of transport intermediates. Such active intermediates have not only been observed by us here and elsewhere (Love et al. 1998), but vesicles and budding profiles on cisternae have been observed by electron microscopy. When these two observations are accounted for, interpretation of the results of the in vitro assay becomes straightforward, and contradictions are removed without the need to invoke hypothetical modes of transport.
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Acknowledgments |
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Submitted: 23 July 1999
Revised: 8 November 1999
Accepted: 9 November 1999
Drs. Lin and Love contributed equally to the manuscript.
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References |
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Balch W.E., Elliott M.M. & Keller D.S.. ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi, J. Biol. Chem., 261, 1986, 14681–14689.
Bannykh S.I. & Balch W.E.. Membrane dynamics at the endoplasmic reticulum Golgi interface, J. Cell Biol., 138, 1997, 1–4.
Beams H.W. & Kessel R.G.. The Golgi apparatusstructure and function, Int. Rev. Cytol., 23, 1968, 209–276.[Medline]
Becker B. & Melkonian M.. The secretory pathway of protistsspatial and functional organization and evolution, Microbiol. Rev, 60, 1996, 697–721.
Belden W.J. & Barlowe C.. Erv25p, a component of COP II-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport, J. Biol. Chem., 271, 1996, 26939–26946.
Bonfanti L., Mironov A.A. Jr., Martinez-Menarguez J.A., Martella O., Fusella A., Baldassarre M., Buccione R., Geuze H.J., Mironov A.A. & Luini A.. Procollagen traverses the Golgi stack without leaving the lumen of cisternaeevidence for cisternal maturation, Cell., 95, 1999, 993–1003.[Medline]
Braell W.A., Balch W.E., Dobbertin D.C. & Rothman J.E.. The glycoprotein that is transported between successive compartments of the Golgi in a cell-free system resides in stacks of cisternae, Cell., 39, 1984, 511–524.[Medline]
Clary D.O., Griff I.C. & Rothman J.E.. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast, Cell., 61, 1990, 709–721.[Medline]
Clermont Y., Rambourg A. & Hermo L.. Connections between the various elements of the cis- and mid-compartments of the Golgi apparatus of early rat spermatids, Anat. Rec., 240, 1994, 469–480.[Medline]
Cole N.B., Ellenberg J., Song J., DiEuliis D. & Lippincott-Schwartz J.. Retrograde transport of Golgi-localized proteins to the ER, J. Cell Biol., 140, 1998, 1–15.
Dominguez M., Dejgaard K., Fullekrug J., Dahan S., Fazel A., Paccaud J.P., Thomas D.Y., Bergeron J.J.M. & Nilsson T.. gp25L/emp24/p24 protein family members of the cis-Golgi network bind COP I and II coatomer, J. Cell Biol., 140, 1998, 751–765.
Dominguez M., Fazel A., Dahan S., Lovell J., Hermo L., Claude A., Melançon P. & Bergeron J.J.M.. Fusogenic domains of Golgi membranes are sequestered into specialized regions of the stack that can be released by mechanical fragmentation, J. Cell Biol., 145, 1999, 673–688.
Donaldson J.G., Kahn R.A., Lippincott-Schwartz J. & Klausner R.D.. Binding of ARF and beta-COP to Golgi membranespossible regulation by a trimeric G protein, Science., 254, 1991, 1197–1199.
Elazar Z., Orci L., Ostermann J., Amherdt M., Tanigawa G. & Rothman J.E.. ADP-ribosylation factor and coatomer couple fusion to vesicle budding, J. Cell Biol., 124, 1994, 415–424.
Farquhar M.G.. Progress in unraveling pathways of Golgi traffic, Annu. Rev. Cell Biol., 1, 1985, 447–488.
Farquhar M.G. & Palade G.E.. The Golgi apparatus100 years of progress and controversy, Trends Cell Biol., 8, 1998, 2–10.[Medline]
Fries E. & Rothman J.E.. Transport of vesicular stomatitis virus glycoprotein in a cell-free extract, Proc. Natl. Acad. Sci. USA., 77, 1980, 3870–3874.
Glick B.S., Elston T. & Oster G.. A cisternal maturation mechanism can explain the asymmetry of the Golgi stack, FEBS Lett., 414, 1997, 177–181.[Medline]
Gottlieb C., Baenziger J. & Kornfeld S.. Deficient uridine diphosphate-N-acetylglucosamine:glycoprotein N-acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins, J. Biol. Chem., 250, 1975, 3303–3309.
Happe S. & Weidman P.. Cell-free transport to distinct Golgi cisternae is compartment specific and ARF-independent, J. Cell Biol., 140, 1998, 511–523.
Ktistakis N.T., Brown H.A., Waters M.G., Sternweis P.C. & Roth M.G.. Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi-coated vesicles, J. Cell Biol., 134, 1996, 295–306.
Ladinsky M.S., Mastronarde D.N., McIntosh J.R., Howell K.E. & Staehelin L.A.. Golgi structure in three dimensionsfunctional insights from the normal rat kidney cell, J. Cell Biol., 144, 1999, 1135–1149.
Lanoix J., Ouwendijk J., Lin C.-C., Stark A., Love H.D., Ostermann J. & Nilsson T.. GTP hydrolysis by arf-1 mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles, EMBO (Eur. Mol. Biol. Organ.) J., 18, 1999, 4935–4948.[Medline]
Letourneur F., Gaynor E.C., Hennecke S., Demolliere C., Duden R. & Emr S.D.. Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum, Cell., 79, 1994, 1199–1207.[Medline]
Lewis M.J. & Pelham H.R.. SNARE-mediated retrograde traffic from the Golgi complex to the endoplasmic reticulum, Cell., 85, 1996, 205–215.[Medline]
Lippincott-Schwartz J., Cole N.B. & Donaldson J.G.. Building a secretory apparatusrole of ARF1/COPI in Golgi biogenesis and maintenance, Histochem. Cell Biol., 109, 1998, 449–462.[Medline]
Love H.D., Lin C.C., Short C.S. & Ostermann J.. Isolation of functional Golgi-derived vesicles with a possible role in retrograde transport, J. Cell Biol., 140, 1998, 541–551.
Lovelock C. & Lucocq J.. Quantitative immuno electron microscopy reveals a2,6 sialyltransferase is concentrated in the central cisternae of rat hepatocyte Golgi apparatus, Eur. J. Cell Biol., 76, 1998, 18–24.[Medline]
Mellman I. & Simons K.. The Golgi complexin vitro veritas?, Cell., 68, 1992, 829–840.[Medline]
Matlin K. & Simons K.. Reduced temperature prevents transfer of a glycoprotein to the cell surface but not terminal glycosylation, Cell., 34, 1983, 233–243.[Medline]
Nilsson T., Pypaert M., Hoe M.H., Slusarewicz P., Berger E.G. & Warren G.. Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells, J. Cell Biol., 120, 1993, 5–13.
Orci L., Glick B.S. & Rothman J.E.. A new type of coated vesicular carrier that appears not to contain clathrinits possible role in protein transport within the Golgi stack, Cell., 46, 1986, 171–184.[Medline]
Orci L., Tagaya M., Amherdt M., Perrelet A., Donaldson J.G., Lippincott-Schwartz J., Klausner R.D. & Rothman J.E.. Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae, Cell., 64, 1991, 1183–1195.[Medline]
Orci L., Stamnes M., Ravazzola M., Amherdt M., Perrelet A., Söllner T.H. & Rothman J.E.. Bidirectional transport by distinct populations of COP I-coated vesicles, Cell., 90, 1997, 335–349.[Medline]
Orci L., Perrelet A. & Rothman J.E.. Vesicles on strings—morphological evidence for progressive transport within the Golgi stack, Proc. Natl. Acad. Sci. USA., 95, 1998, 2279–2283.
Ostermann J., Orci L., Tani K., Amherdt M., Ravazzola M., Elazar Z. & Rothman J.E.. Stepwise assembly of functionally active transport vesicles, Cell., 75, 1993, 1015–1025.[Medline]
Pelham H.R.B.. The dynamic organisation of the secretory pathway, Cell Struct. Funct., 21, 1996, 413–419.[Medline]
Peter F., Plutner H., Zhu H., Kreis T.E. & Balch W.E.. Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro, J. Cell Biol., 122, 1993, 1155–1167.
Pfanner N., Orci L., Glick B.S., Amherdt M., Arden S.R., Malhotra V. & Rothman J.E.. Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae, Cell., 59, 1989, 95–102.[Medline]
Presley J.F., Cole N.B., Schroer T.A., Hirschberg K., Zaal K.J.M. & Lippincott-Schwartz J.. ER-to-Golgi transport visualized in living cells, Nature., 389, 1997, 81–84.[Medline]
Rambourg A., Clermont Y. & Képès F.. Modulation of the Golgi apparatus in Saccharomyces cerevisiae sec7 mutants as seen by three-dimensional electron microscopy, Anat. Rec., 237, 1993, 441–452.[Medline]
Rojo M., Pepperkok R., Emery G., Kellner R., Stang E., Parton R.G. & Gruenberg J.. Involvement of the transmembrane protein p23 in biosynthetic protein transport, J. Cell Biol., 139, 1997, 1119–1135.
Saraste J. & Kuismanen E.. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus membrane glycoproteins to the cell surface, Cell., 38, 1984, 535–549.[Medline]
Scales S.J., Pepperkok R. & Kreis T.E.. Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COP II and COP I, Cell., 90, 1997, 1137–1148.[Medline]
Schimmoller F., Singer-Kruger B., Schroder S., Kruger U., Barlowe C. & Riezman H.. The absence of Emp24p, a component of ER-derived COP II-coated vesicles, causes a defect in transport of selected proteins to the Golgi, EMBO (Eur. Mol. Biol. Organ.) J., 14, 1995, 1329–1339.[Medline]
Shima D.T., Cabrera-Poch N., Pepperkok R. & Warren G.. An ordered inheritance strategy for the Golgi apparatusvisualization of mitotic disassembly reveals a role for the mitotic spindle, J. Cell Biol., 141, 1998, 955–966.
Sönnichsen B., Watson R., Clausen H., Misteli T. & Warren G.. Sorting by COP I–coated vesicles under interphase and mitotic conditions, J. Cell Biol., 134, 1996, 1411–1425.
Sönnichsen B., Lowe M., Levine T., Jämsä E., Dirac-Svejstrup B. & Warren G.. A role for giantin in docking COP I vesicles to Golgi membranes, J. Cell Biol., 140, 1998, 1013–1021.
Stamnes M.A., Craighead M.W., Hoe M.H., Lampen N., Geromanos S., Tempst P. & Rothman J.E.. An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding, Proc. Natl. Acad. Sci. USA., 92, 1995, 8011–8015.
Storrie B., White J., Rottger S., Stelzer E.H., Suganuma T. & Nilsson T.. Recycling of Golgi-resident glycosyltransferases through the ER reveals a novel pathway and provides an explanation for nocodazole-induced Golgi scattering, J. Cell Biol., 143, 1998, 1505–1521.
Taylor T.C., Kanstein M., Weidman P. & Melançon P.. Cytosolic ARFs are required for vesicle formation but not for cell-free intra-Golgi transportevidence for coated vesicle-independent transport, Mol. Biol. Cell., 5, 1994, 237–252.[Abstract]
Velasco A., Hendricks L., Moremen K.W., Tulsiani D.R., Touster O. & Farquhar M.G.. Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II, J. Cell Biol., 122, 1993, 39–51.
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